Effects of growth factors on multipotent bone marrow mesenchymal stromal cells

Multipotent bone marrow mesenchymal stromal cells are progenitors of various cell types capable of long-term self-renewal. These cells are an adequate model for studying the most important problems in cell biology, such as self-renewal of stem cells and regulation of their differentiation. Moreover, these cells are a promising resource for regenerative medicine. In this context, isolation of the earliest multipotent mesenchymal stromal cells, their in vitro maintenance in an undifferentiated state, and stimulation of their differentiation in a desired direction appear to be most important. To successfully use the multipotent mesenchymal stromal cells both in fundamental studies and in therapy, it is necessary to modify and standardize the composition of culture medium, replacing blood serum with certain growth factors. These factors have influence on the proliferation and differentiation of most cell types, including multipotent mesenchymal stromal cells. This paper is a review of available data concerning the effects of some growth factors on the multipotent mesenchymal stromal cells of the bone marrow.     

The Cannabinoid Receptor Type 2 as Mediator of Mesenchymal Stromal Cell Immunosuppressive Properties

Mesenchymal stromal cells are non-hematopoietic, multipotent progenitor cells producing cytokines, chemokines, and extracellular matrix proteins that support hematopoietic stem cell survival and engraftment, influence immune effector cell development, maturation, and function, and inhibit alloreactive T-cell responses. The immunosuppressive properties of human mesenchymal stromal cells have attracted much attention from immunologists, stem cell biologists and clinicians.Recently, the presence of the endocannabinoid system in hematopoietic and neural stem cells has been demonstrated. Endocannabinoids, mainly acting through the cannabinoid receptor subtype 2, are able to modulate cytokine release and to act as immunosuppressant when added to activated T lymphocytes.In the present study, we have investigated, through a multidisciplinary approach, the involvement of the endocannabinoids in migration, viability and cytokine release of human mesenchymal stromal cells.We show, for the first time, that cultures of human mesenchymal stromal cells express all of the components of the endocannabinoid system, suggesting a potential role for the cannabinoid CB2 receptor as a mediator of anti-inflammatory properties of human mesenchymal stromal cells, as well as of their survival pathways and their capability to home and migrate towards endocannabinoid sources.     

Mesenchymal stromal progenitor cells: general characteristics and functional state in low oxygen tension

The promising use of multipotent mesenchymal stromal cells (MSC) in regenerative technologies accounts for necessity of detailed study and analysis of their physiology. Along with searching for MSC specific immunophenotype markers and factors that affect MSC heterogeneity, proliferative activity, self-renewal capacity and differentiation potential, progenitor, the cells functioning in low oxygen tension present one the open-ended problems. The specific response of progenitor cells to hypoxia seems to depend on many factors including their commitment and differentiation status, tissue type as well as the nature of the hypoxic impact. Interestingly, it was shown that low oxygen tension can be used as a factor regulating MSC proliferation, viability and differentiation.     

Differentiation Capacity toward Mesenchymal Cell Lineages of Bone Marrow Stromal Cells Established from Temperature-Sensitive SV40 T-Antigen Gene Transgenic Mouse

Stromal cell lines were established from bone marrow of temperature-sensitive T-antigen gene transgenic mice. These stromal cell lines consisted of fibroblasts, endothelial cells, and preadipocytes. We found that these stromal cell lines exhibited phenotypic changes depending on the inactivation of T-antigen and growth condition; one preadipocyte line was induced toward adipocytes and osteogenic cells, and several preadipocyte and endothelial cell lines were induced toward muscle cells and adipocytes. Some cell lines showed bipotential characters. These results indicated that stromal cells consisting of bone marrow hematopoietic microenvironment are derived from multipotent mesenchymal stem cells.     

Postnatal stem/progenitor cells derived from the dental pulp of adult chimpanzee

Background  

Chimpanzee dental pulp stem/stromal cells (ChDPSCs) are very similar to human bone marrow derived mesenchymal stem/stromal cells (hBMSCs) as demonstrated by the expression pattern of cell surface markers and their multipotent differentiation capability.     

Comparative characterization of mesenchymal bone marrow stromal cells at early and late stages of culturing

The mesenchymal stromal cell is a multipotent precursor of osteoblasts, adipocytes, and some other cell types. In this study, a comparative analysis of cultured mesenchymal stromal cells from the rat bone marrow at the early and late stages of subculturing has been performed using molecular genetic and cytological methods. The culture has undergone 11 passages during 140 days. Upon long-term culturing, the mesenchymal stromal cells have proved to lose their potential for adipogenic differentiation but preserve the potential for osteogenesis. Morphological characters typical of osteogenic differentiation can be observed at the earlier stages of culturing (passages 1–4) but disappear at later stages (passages 9–11), despite mineralization of the extracellular matrix and the expression of osteogenic differentiation markers. A comparative analysis of the proliferation potential of stromal cells has shown that differences in the period of cell population doubling at the early and later stages of culturing are insignificant. An almost complete arrest of cell growth has been observed in the middle of the culture period (passages 5 and 6).     

The impact of oxygen in physiological regulation of human multipotent mesenchymal cell functions

Significant progress in studying cellular mechanisms of tissue homeostasis and physiological remodeling has been made in recent decades. Undifferentiated cells, such as multipotent mesenchymal stromal (stem) cells (MMSCs), play an important role in these processes. MMSCs were found in practically all organs occupying specific tissue niches associated with the perivascular spaces. The main characteristic of MMSCs is their ability, on the one hand, to provide structural integrity of tissues and, on the other hand, to respond to paracrine stimuli and migrate to damaged target tissues, which promotes tissue reparation. A low partial oxygen tension is the main feature of the physiological and regeneration microenvironment, which may significantly modify stromal cell properties. This review analyzes the recent data on MMSC tissue niches in terms of the integration of these cells into a comprehensive system of physiological and reparative tissue remodeling and the role of partial oxygen pressure in the fulfillment of the MMSC potential.     

Purmorphamine enhances osteogenic activity of human osteoblasts derived from bone marrow mesenchymal cells

Purmorphamine is a novel small molecule with osteogenesis-inducing activity in multipotent mesenchymal progenitor cells, but there has been no evaluation of its effect on human cells to date. The aim of this study was to investigate the induction of osteogenic activity by purmorphamine in human osteoblasts differentiated from bone marrow mesenchymal cells. Cells were cultured in 24-well plates at a density of 2x10(4)/well in medium containing 1, 2 or 3 microM purmorphamine, or vehicle. At 7, 14 and 21 days, cell proliferation, viability, and alkaline phosphatase (ALP) activity were evaluated. Bone-like nodule formation was evaluated at 21 days. Purmorphamine did not affect cell proliferation or viability, but increased ALP activity and bone-like nodule formation. These results indicate that events related to osteoblast differentiation, including increased ALP activity and bone-like nodule formation, are enhanced by purmorphamine.     

Human mesenchymal progenitor cells derived from alveolar bone and human bone marrow stromal cells: a comparative study

The aim of the present study was to evaluate the potential of intraoral harvested alveolar bone as an alternative source of multipotent mesenchymal stromal cells for future applications in oral and maxillofacial tissue engineering. Explant cultures were established from 20 alveolar bone samples harvested from the oblique line immediately before wisdom tooth removal. Morphology and proliferation characteristics of the in vitro expanded cells, referred to as human alveolar bone-derived cells (hABDCs), were studied using phase-contrast microscopy. Immunocytochemical analysis of their surface marker expression was conducted using monoclonal antibodies defining mesenchymal stromal cells. To evaluate their multilineage differentiation potential, hABDCs were induced to differentiate along the osteogenic, adipogenic, and chondrogenic lineage and compared to bone marrow mesenchymal stromal cells (hBMSCs) on mRNA and protein levels applying RT-PCR and cytochemical staining methods. hABDCs showed typical morphological characteristics comparable to those of hBMSCs such as being mononuclear, fibroblast-like, spindle-shaped, and plastic adherent. Immunophenotypically, cells were positive for CD105, CD90, and CD73 while negative for CD45, CD34, CD14, CD79α, and HLA-DR surface molecules, indicating an antigen expression pattern considered typical for multipotent mesenchymal stromal cells. As evidenced by RT-PCR and cytochemistry, hABDCs showed multilineage differentiation and similar chondrogenic and osteogenic differentiation potentials when compared to hBMSCs. Our findings demonstrate that human alveolar bone contains mesenchymal progenitor cells that can be isolated and expanded in vitro and are capable of trilineage differentiation, providing a reservoir of multipotent mesenchymal cells from an easily accessible tissue source.     

Long-term expansion of multipotent mesenchymal stromal cells under reduced oxygen tension

It has been shown that a decrease in oxygen tension during cultivation of multipotent mesenchymal stromal cells (MMSCs) caused a short-term decline in the proportion of CD73⁺ cells in the population, with no effect on the number of cells expressing the other constitutive surface markers (CD90, CD105). The heterogeneity of the cell population declined: large spread cells disappeared. The proliferative activity of MMSCs significantly increased and remained stable under conditions close to tissue oxygen levels (5% O₂). At lower oxygen concentration, it is gradually reduced from the third to fourth passages. The increase in proliferative activity was not accompanied by increased telomerase gene expression, which indicated that no cell transformation had occurred. Global gene expression analysis of MMSC gene expression revealed changes in expression of cyclins (CCND2, PCNA), regulatory subunit cyclin-dependent kinase (CKS2), and an inhibitor of cyclin-dependent kinases 4 and 6 (CDKN2C) regulating the cell cycle, which probably facilitated the proliferative activity of cells at lower oxygen tension.     

Hypoxia-Controlled EphA3 Marks a Human Endometrium-Derived Multipotent Mesenchymal Stromal Cell that Supports Vascular Growth

Eph and ephrin proteins are essential cell guidance cues that orchestrate cell navigation and control cell-cell interactions during developmental tissue patterning, organogenesis and vasculogenesis. They have been extensively studied in animal models of embryogenesis and adult tissue regeneration, but less is known about their expression and function during human tissue and organ regeneration. We discovered the hypoxia inducible factor (HIF)-1α-controlled expression of EphA3, an Eph family member with critical functions during human tumour progression, in the vascularised tissue of regenerating human endometrium and on isolated human endometrial multipotent mesenchymal stromal cells (eMSCs), but not in other highly vascularised human organs. EphA3 affinity-isolation from human biopsy tissue yielded multipotent CD29⁺/CD73⁺/CD90⁺/CD146⁺ eMSCs that can be clonally propagated and respond to EphA3 agonists with EphA3 phosphorylation, cell contraction, cell-cell segregation and directed cell migration. EphA3 silencing significantly inhibited the ability of transplanted eMSCs to support neovascularisation in immunocompromised mice. In accord with established roles of Eph receptors in mediating interactions between endothelial and perivascular stromal cells during mouse development, our findings suggest that HIF-1α-controlled expression of EphA3 on human MSCs functions during the hypoxia-initiated early stages of adult blood vessel formation.     

Characteristics of human lipoaspirate-isolated mesenchymal stromal cells cultivated under lower oxygen tension

The effect of reduced oxygen concentration in the gas phase on the proliferation, viability, and immunophenotype of human mesenchymal stromal cells isolated from lipoaspirate (lMSC) has been investigated. It was shown that the proliferation activity of cells under hypoxic conditions (5% O₂) was, on average, 2.9 times higher than those cultivated under routine (normoxic) (20% O₂) conditions. Decreased oxygen level in the culture medium did not cause any change in lMSC viability or immunophenotype. Thus, the permanent cultivation of lMSC in medium with a lower oxygen tension may be an efficient approach to obtaining a higher mass of cells that maintain their characteristics over a shorter period of time, which is a requirement for regenerative medicine.     

Human mesenchymal and murine stromal cells support human lympho-myeloid progenitor expansion but not maintenance of multipotent haematopoietic stem and progenitor cells

A major goal in haematopoietic stem cell (HSC) research is to define conditions for the expansion of HSCs or multipotent progenitor cells (MPPs). Since human HSCs/MPPs cannot be isolated, NOD/SCID repopulating cell (SRC) assays emerged as the standard for the quantification of very primitive haematopoietic cell. However, in addition to HSCs/MPPs, lympho-myeloid primed progenitors (LMPPs) were recently found to contain SRC activities, challenging this assay as clear HSC/MPP readout. Because our revised model of human haematopoiesis predicts that HSCs/MPPs can be identified as CD133⁺CD34⁺ cells containing erythroid potentials, we investigated the potential of human mesenchymal and conventional murine stromal cells to support expansion of HSCs/MPPs. Even though all stromal cells supported expansion of CD133⁺CD34⁺ progenitors with long-term myeloid and long-term lymphoid potentials, erythroid potentials were exclusively found within erythro-myeloid CD133^lowCD34⁺ cell fractions. Thus, our data demonstrate that against the prevailing assumption co-cultures on human mesenchymal and murine stromal cells neither promote expansion nor maintenance of HSCs and MPPs.     

Tonsil-derived mesenchymal progenitor cells acquire a follicular dendritic cell phenotype under cytokine stimulation

Tonsils comprise part of the mucosal immune system and contain lymphocytes, macrophages, and follicular dendritic cells (FDCs). FDCs are located in the B cell area of the follicles of secondary lymphoid organs, such as the spleen, tonsils, or lymph nodes, and they trap and retain immune complexes on their surfaces to regulate B cell activation and maturation. Stromal cells from the palatine tonsils are often used for FDC in vitro studies, and it has been reported that human palatine tonsils may be a good source of multipotent mesenchymal cells. Therefore, we assessed whether tonsil-derived mesenchymal stromal cells could differentiate into a FDC-like phenotype. We discovered that stromal cells isolated from human tonsils not only had the potential to differentiate into various cell types of mesenchymal origin, but they also could differentiate into FDC-like cells under cytokine stimulation in vitro.     

The cultivation of bone marrow cells and various cell lines on polymer films

The cultivation of multipotent mesenchymal stromal bone marrow cells (BM-MSC) and A-431, MDCK, Vero, 3T3 and Hep-G₂ lines of cells was performed in DMEM medium using polyvinyl alcohol (PVA) polymer films as scaffolds. PVA was modified by covalent linking of various hydrophobic fatty acids. Different cell types exhibited different growth intensity on these films, which, however, in most cases was comparable with control cultivations on plastics.     

Short-term exposure of multipotent stromal cells to low oxygen increases their expression of CX3CR1 and CXCR4 and their engraftment in vivo

The ability of stem/progenitor cells to migrate and engraft into host tissues is key to their potential use in gene and cell therapy. Among the cells of interest are the adherent cells from bone marrow, referred to as mesenchymal stem cells or multipotent stromal cells (MSC). Since the bone marrow environment is hypoxic, with oxygen tensions ranging from 1% to 7%, we decided to test whether hypoxia can upregulate chemokine receptors and enhance the ability of human MSCs to engraft in vivo. Short-term exposure of MSCs to 1% oxygen increased expression of the chemokine receptors CX3CR1and CXCR4, both as mRNA and as protein. After 1-day exposure to low oxygen, MSCs increased in vitro migration in response to the fractalkine and SDF-1alpha in a dose dependent manner. Blocking antibodies for the chemokine receptors significantly decreased the migration. Xenotypic grafting into early chick embryos demonstrated cells from hypoxic cultures engrafted more efficiently than cells from normoxic cultures and generated a variety of cell types in host tissues. The results suggest that short-term culture of MSCs under hypoxic conditions may provide a general method of enhancing their engraftment in vivo into a variety of tissues.     

Human subcutaneous adipose tissue subjected to cold shock as a source of viable cellular population with characteristics of multipotent mesenchymal stromal cells

Cellular population with characteristics of multipotent mesenchymal stromal cells (MMSCs) was isolated from subcutaneous adipose tissue frozen without any cryoprotectant at -70 degrees C. Under critical for the adipose tissue condition, the cells retained their viability in vitro and ability of adhesion to plastic. Cellular population was homogeneous and represented by small cells (d - 7 microm) with fibroblast-like morphology. Cells were positively stained with Abs for the Abs: CD29, CD44, CD49a, b, d, CD73, CD90, CD105, CD166, HLA ABC. Cells were negative for CD34, CD45--markers of hematopoietic cells, CD31--marker of endothelial cells, Stro-1, as well as for HLA DR, DP, DQ (flow cytometer analysis). Being induced to differentiate in vitro, the cells were able to differentiate into cells similar to cells of bone, adipose and cartilage tissue. Karyological assay of the cells isolated from human adipose tissue subjected to cold shock revealed diploid set of chromosomes, 46, XX, without aneuploidy and structural reconstructions of chromosomes. Thus, it has been established that, under extreme condition for the organism, the population of cells with a phenotype similar to miltipotent mesenchymal stromal cells is preserved in subcutaneous adipose tissue.     

Effect of oestradiol on cytokine production in immortalized human marrow stromal cell lines.

Oestrogen deficiency enhances bone osteoclastogenesis and bone resorption. Evidence of cooperation between stromal cells and osteoclast precursors in mice suggests that oestradiol acts by regulating cytokine release from stromal cells. Bone marrow stroma contains multipotent progenitors that give rise to many mesenchymal lineages, including osteoblasts that may regulate osteoclast differentiation. We immortalized and characterized six human bone marrow stromal cell lines (presence of Stro1, secretion of alkaline phosphatase, osteocalcin, formation of lipid droplets, and presence of alpha and beta oestrogen receptors). The response of cytokines to oestradiol was then evaluated in vitro, as were the phorbol myristate acetate (PMA)-stimulated cytokine levels. Cells had the characteristics of undifferentiated stromal cells (Stro1+, RANK-L+), and expressed alpha-oestrogen receptors. The osteoblast phenotype (amounts of alkaline phosphatase and osteocalcin) was weak and there was a poor capacity to differentiate into adipocytes. These cell lines did not respond to oestradiol by producing interleukin 6 (IL-6), IL-1 or tumour necrosis factor alpha (TNF-alpha) either constitutively or after stimulation with PMA. Moreover, RANK-L and osteoprotegerin expressions were not regulated by oestradiol in vitro. Thus, modulation of these cytokines by stromal cells do not appear to be the mechanism by which oestradiol regulates bone resorption in humans.     

Expression of senescence-associated genes in multipotent mesenchymal stromal cells during long-term cultivation at various hypoxic levels

The expression of several genes which functions are associated with cellular senescence was analyzed in multipotent mesenchymal stromal cells during long-term cultivation at different oxygen levels (20, 5, and 1%) using the RT² Profiler? PCR Array Human Cellular Senescence system (Qiagen, United States). It was established that replicative senescence processes develop most actively in the cells cultured under the standard conditions (20% O₂). The most significant changes were observed in the expression of CCND1, ID1, IGF1, PIK3CA, and SERPINE1 genes.