Mesenchymal stem cells (MSCs) represent a heterogeneous group of multipotent stem cells that could be found in various somatic tissues. MSCs are defined by molecular and functional features including spindle-shape morphology, adherence to plastic surfaces, expression of specific surface markers and differentiation potential to chondrocytes, adipocytes and osteocytes. The surface markers were proposed to affect the differentiation potential of MSCs by a limited number of studies. Endoglin (CD105) is defined to be a significant marker for osteogenic and chondrogenic differentiation ability of MSCs. Low CD105 expression is associated with increased osteogenic potential while high CD105 expression is correlated with strong chondrogenic potential. Myrtucommulone-A (MC-A) is an active compound with various biological effects on different cell types but its effect on MSC differentiation has not been described yet. In the present study we aimed at investigating the longterm effects of MC-A on hMSCs. MC-A-treatment reduced CD105 expression in distinct human mesenchymal stem cell (hMSC) lines and gave rise to CD105(low) population but did not change CD44, CD90 or CD73 expression. The decrease in CD105 expression reduced the chondrogenic potential of hMSCs subsequently while adipogenic or osteogenic differentiation was not affected dramatically. MC-A-treatment also suppressed the NF-κB p65 activation which might be responsible for the reduced chondrogenic potential. Our findings suggest thatMC-Acould be used to enrichCD105(low)hMSCs without the need for cell sorting or changing culture conditions which could be utilised in targeted differentiation studies.
Adipocytes ; Chondrocytes ; Humans ; Mesenchymal Stromal Cells* ; Multipotent Stem Cells ; Osteocytes ; Plastics

Adult stem cells are drawing more and more attention due to the potential application in degenerative medicine without posing any moral problem. There is growing evidence showing that the human amnion contains various types of adult stem cell. Since amniotic tissue is readily available, it has the potential to be an important source of regenerative medicine material. In this study we tried to find multipotent adult stem cells in human amnion. We isolated stem cells from amniotic mesenchymal cells by limiting dilution assay. Similar to bone marrow derived mesenchymal stem cells, these cells displayed a fibroblast like appearance. They were positive for CD105, CD29, CD44, negative for haematopoietic (GlyA, CD31, CD34, CD45) and epithelial cell (pan-CK) markers. These stem cells had the potential to differentiate not only into osteogenic, adipogenic and endothelial lineages, but also hepatocyte-like cells and neural cells at the single-cell level depending on the culture conditions. They had the capacity for self-renewal and multilineage differentiation even after being expanded for more than 30 population doublings in vitro. So they may be an ideal stem cell source for inherited or degenerative diseases treatment.
Adult Stem Cells ; cytology ; Amnion ; cytology ; Cell Differentiation ; physiology ; Humans ; Mesenchymal Stromal Cells ; cytology ; Multipotent Stem Cells ; cytology

BACKGROUND AND OBJECTIVES: Mesenchymal stem cells (MSCs) are multipotent stem cells that can be isolated from umbilical cords and are therapeutically used because of their ability to differentiate into various types of cells, in addition to their immunosuppressive and anti-inflammatory properties. Fetal bovine serum (FBS), considered as the standard additive when isolating and culturing MSCs, has a major limitation related to its animal origin. Here, we employed a simple and economically efficient protocol to isolate MSCs from human umbilical cord tissues without using digestive enzymes and replacing FBS with umbilical cord blood serum (CBS). METHODS AND RESULTS: MSCs were isolated by culturing umbilical cord pieces in CBS or FBS supplemented media. Expansion and proliferation kinetics of cells isolated by explant method in the presence of either FBS or CBS were measured, with morphology and multi-differentiation potential of expanded cells characterized by flow cytometry, RT-PCR, and immunofluorescence. MSCs maintained morphology, immunophenotyping, multi-differentiation potential, and self-renewal ability, with better proliferation rates for cells cultured in CBS compared to FBS supplement media. CONCLUSIONS: We here present a simple, reliable and efficient method to isolate MSCs from umbilical cord tissues, where cells maintained proliferation, differentiation potential and immunophenotyping properties and could be efficiently expanded for clinical applications.
Animals ; Fetal Blood* ; Flow Cytometry ; Fluorescent Antibody Technique ; Humans* ; Immunophenotyping ; Kinetics ; Mesenchymal Stromal Cells* ; Methods* ; Multipotent Stem Cells ; Umbilical Cord*

Human mesenchymal stem cells (hMSCs) are multipotent stem cells that can differentiate into several mesenchymal lineage cells. In this study, we established an efficient method for gene delivery into these cells. Non-viral transfection reagents that were commercially available yielded 5% efficiency. In contrast, a retroviral vector yielded more than 46% transduction, which was further increased to 90% by repetitive infection. Retroviral transduction did not alter the multipotency of hMSCs. Thus, the cells retained the potential to differentiate into adipogenic, chondrogenic, or osteogenic lineages. The conditions established in this study will contribute to development of trans-differentiation methods of hMSCs into non-mesodermal lineage cells and thereby facilitate their possible use as vehicles for autologous transplantation in both cell and gene therapy for various diseases.
Autografts ; Genetic Therapy ; Humans* ; Indicators and Reagents ; Mesenchymal Stromal Cells* ; Multipotent Stem Cells ; Retroviridae ; Transfection ; Transplantation, Autologous ; Zidovudine*

BACKGROUND: Mesenchymal stromal cells (MSCs) are multipotent stem cells that can differentiate into several cell types. In addition, many studies have shown that MSCs modulate the immune response. However, little information is currently available regarding the maintenance of immunomodulatory characteristics of MSCs through passages. Therefore, we investigated and compared cytokine and gene expression levels from adipose (AD) and bone marrow (BM)-derived MSCs relevant to immune modulation from early to late passages. METHODS: MSC immunophenotype, growth characteristics, cytokine expressions, and gene expressions were analyzed. RESULTS: AD-MSCs and BM-MSCs had similar cell morphologies and surface marker expressions from passage 4 to passage 10. Cytokines secreted by AD-MSCs and BM-MSCs were similar from early to late passages. AD-MSCs and BM-MSCs showed similar immunomodulatory properties in terms of cytokine secretion levels. However, the gene expressions of tumor necrosis factor-stimulated gene (TSG)-6 and human leukocyte antigen (HLA)-G were decreased and gene expressions of galectin-1 and -3 were increased in both AD- and BM-MSCs with repeated passages. CONCLUSION: Our study showed that the immunophenotype and expression of immunomodulation-related cytokines of AD-MSCs and BM-MSCs immunomodulation through the passages were not significantly different, even though the gene expressions of both MSCs were different.
Bone Marrow ; Cytokines* ; Galectin 1 ; Gene Expression ; Humans* ; Immunomodulation ; Leukocytes ; Mesenchymal Stromal Cells* ; Multipotent Stem Cells ; Necrosis

A series of studies have demonstrated that bone marrow mesenchymal stem cells (MSCs) are attractive candidates for cell and gene therapies, because they are readily obtained and multipotentially differentiated. Then homogeneous MSC cultures in vitro with more rapidly self-renewing ability and multipotential differentiation will accelerate and improve their progress in clinical application. Colter et al. found that early colonies contain a third kind of cells very small round cells that rapidly self-renew, besides spindle-shaped cells and large flat cells, called RS cells. RS cells are characterized by their extremely small size, rapid rate of replication, and enhanced potential for multilineage differentiation. Moreover, they can be distinguished from more mature cells in the same cultures by a series of surface epitopes and expressed proteins. Therefore, the results raise the possibility that RS cells may have the greatest potential for long-term engraftment and differentiation in vivo.
Bone Marrow Cells ; cytology ; Cell Differentiation ; Cells, Cultured ; Humans ; Mesenchymal Stromal Cells ; cytology ; Multipotent Stem Cells ; classification ; cytology ; Stromal Cells ; cytology

Human mesenchymal stem cells (hMSCs) are multipotent stem cells that can differentiate into several mesenchymal lineage cells. In this study, we established conditions that allowed a long term expansion of hMSCs. To search for the optimum culture condition, growth rates of hMSCs were measured in the presence of several growth factors. Hepatic growth factor (HGF) and leukemia inhibitory factor (LIF) did not facilitate proliferation of hMSCs. In contrast, basic fibroblast growth factor (bFGF) effectively promoted growth of the cells in vitro by 3 fold. The growth stimulatory effect of bFGF was dependent on the concentration. The adipogenic potential was dramatically decreased in hMSCs isolated from an aged donor whereas osteogenic potential was minimally decreased. Addition of bFGF resumed the adipogenic and osteogenic differentiation potential. Thus, the cells that expanded in the presence of bFGF retained the potential to differentiate into adipogenic, chondrogenic, or osteogenic lineage cells. MSCs could be expanded for at least 8 passages with bFGF and the resulting cells retained the normal karyotype. The cells were positive for CD9, CD13, CD15, CD90, CD137, and CD140b; but negative for CD14, CD34, and CD45. Importantly, the cells were found to express a neural stem cell marker, nestin, and a neuronal marker, beta-tubulin III. The results suggest that bFGF promote proliferation while maintaining multi-lineage differentiation potency of hMSCs. Finally, we suggest that it is critical to identify novel markers other than nestin or beta-tubulin III to monitor acquisition of neuronal phenotypes by hMSCs.
Fibroblast Growth Factor 2* ; Humans* ; Intercellular Signaling Peptides and Proteins ; Karyotype ; Leukemia Inhibitory Factor ; Mesenchymal Stromal Cells* ; Multipotent Stem Cells ; Nestin ; Neural Stem Cells ; Neurons ; Phenotype ; Tissue Donors ; Tubulin

This study was aimed to investigate if human heart harbored a population of primitive undifferentiated cells with the characteristics of MPC. Cells were isolated from human fetal heart and were cultured under conditions appropriate for bone marrow-derived MPCs. Their morphology, phenotypes and functions were tested by methods developed for MPC from other sources. The results showed that morphologically, cells were spindle shaped and resembled fibroblasts. In their undifferentiated state, cells were CD73, CD105, CD29, CD44, HLA-ABC, CD166 positive and CD45, CD34, CD86, HLA-DR negative. When cultured in adipogenic, osteogenic or chondrogenic media, cells differentiated into adipocytes, osteocytes and chondrocytes respectively. They could be extensively expanded in vitro and exhibited very low immunogenicity as evaluated by T cell proliferation assays. It is concluded that cells isolated from fetal heart possess similarity to their adult and fetal bone marrow counterparts in morphologic, immunophenotypic, and functional characteristics.
Bone Marrow Cells ; cytology ; Cell Adhesion ; Cell Differentiation ; Cells, Cultured ; Fetal Heart ; cytology ; Fetus ; Humans ; Mesenchymal Stromal Cells ; cytology ; immunology ; Multipotent Stem Cells ; physiology

Animals ; Cell Culture Techniques ; methods ; Cell Differentiation ; Cell Separation ; methods ; Cells, Cultured ; Embryonic Stem Cells ; cytology ; Hepatocytes ; cytology ; Humans ; Liver Failure ; therapy ; Liver, Artificial ; Mesenchymal Stromal Cells ; cytology ; Multipotent Stem Cells ; cytology ; Stem Cell Transplantation ; Stem Cells ; cytology

OBJECTIVETo investigate the biological features of human decidua basalis-derived mesenchymal stem cells (PDB-MSCs) in vitro and identify their capacity of multilineage differentiation.

METHODSPDB-MSCs were harvested from the decidua basalis of term placental by enzymatic digestion and density gradient centrifugation, and the growth characteristics and morphological changes of the MSCs were observed by inverted microscope. The proliferative ability of the cells was assessed by Cell Counting Kit-8. The cell cycle and expressions of the surface markers (CD29, CD44, CD73, CD90, CD34, CD45, and CD14) of the MSCs were identified by flow cytometry. Multilineage differentiation capacity of the cells was tested by inducing their differentiation toward osteoblasts, adipocytes and chondroblasts in vitro.

RESULTSMSCs isolated from human decidua basalis of term placental exhibited a morphology similar to that of bone marrow-derived MSCs, and grew into colonies in in vitro culture, where the cells proliferated rapidly after passage with a cell doubling time of 2.21∓0.21 days. More than 70% of the cells stayed in the resting stage (G(0)/G(1)) and showed positivity for CD29, CD44, CD73 and CD90, but not for CD14, CD34 or CD45. After induction, the cells showed positive results of alizarin red staining, oil red O staining and Alcian blue staining.

CONCLUSIONHuman decidua basalis contains a rich source of MSCs, which can be easily isolated and cultured without affecting their capacity of multilineage differentiation. The PDB-MSCs may have the potential as a novel source of stem cells.

Cell Differentiation ; physiology ; Cell Separation ; Cells, Cultured ; Decidua ; cytology ; Female ; Humans ; Mesenchymal Stromal Cells ; cytology ; Multipotent Stem Cells ; cytology ; Placenta ; cytology ; Pregnancy