Low physiologic oxygen tensions reduce proliferation and differentiation of human multipotent mesenchymal stromal cells

Background

Human multipotent mesenchymal stromal cells (MSC) can be isolated from various tissues including bone marrow. Here, MSC participate as bone lining cells in the formation of the hematopoietic stem cell niche. In this compartment, the oxygen tension is low and oxygen partial pressure is estimated to range from 1% to 7%. We analyzed the effect of low oxygen tensions on human MSC cultured with platelet-lysate supplemented media and assessed proliferation, morphology, chromosomal stability, immunophenotype and plasticity.

Results

After transferring MSC from atmospheric oxygen levels of 21% to 1%, HIF-1α expression was induced, indicating efficient oxygen reduction. Simultaneously, MSC exhibited a significantly different morphology with shorter extensions and broader cell bodies. MSC did not proliferate as rapidly as under 21% oxygen and accumulated in G₁ phase. The immunophenotype, however, was unaffected. Hypoxic stress as well as free oxygen radicals may affect chromosomal stability. However, no chromosomal abnormalities in human MSC under either culture condition were detected using high-resolution matrix-based comparative genomic hybridization. Reduced oxygen tension severely impaired adipogenic and osteogenic differentiation of human MSC. Elevation of oxygen from 1% to 3% restored osteogenic differentiation.

Conclusion

Physiologic oxygen tension during in vitro culture of human MSC slows down cell cycle progression and differentiation. Under physiological conditions this may keep a proportion of MSC in a resting state. Further studies are needed to analyze these aspects of MSC in tissue regeneration.     

Multi-lineage differentiation of human mesenchymal stromal cells on the biophysical microenvironment of cell-derived matrix

We obtained fibroblast- (FDM) and preosteoblast- (PDM) derived matrices in vitro from their respective cells. Our hypothesis was that these naturally occurring cell-derived matrices (CDMs) would provide a better microenvironment for the multi-lineage differentiation of human mesenchymal stromal cells (hMSCs) than those based on traditional single-protein-based platforms. Cells cultured for 5–6 days were decellularized with detergents and enzymes. The resulting matrices showed a fibrillar surface texture. Under osteogenic conditions, human bone-marrow-derived stromal cells (HS-5) exhibited higher amounts of both mineralized nodule formation and alkaline phosphatase (ALP) expression than those cultured on plastic or gelatin. Osteogenic markers (Col I, osteopontin, and cbfa1) and ALP activity from cells cultured on PDM were notably upregulated at 4 weeks. The use of FDM significantly improved the cellular expression of chondrogenic markers (Sox 9 and Col II), while downregulating that of Col I at 4 weeks. Both CDMs were more effective in inducing cellular synthesis of glycosaminoglycan content than control substrates. We also investigated the effect of matrix surface texture on hMSC (PT-2501) differentiation; soluble matrix (S-matrix)-coated substrates exhibited a localized fibronectin (FN) alignment, whereas natural matrix (N-matrix)-coated substrates preserved the naturally formed FN fibrillar alignment. hMSCs cultured for 4 weeks on N-matrices under osteogenic or chondrogenic conditions deposited a greater amount of calcium and proteoglycan than those cultured on S-matrices as assessed by von Kossa and Safranin O staining. In contrast to the expression levels of lineage-specific markers for cells cultured on gelatin, FN, or S-matrices, those cultured on N-matrices yielded highly upregulated levels. This study demonstrates not only the capacity of CDM for being an effective inductive template for the multi-lineage differentiation of hMSCs, but also the critical biophysical role that the matrix fibrillar texture itself plays on the induction of stem cell differentiation.     

Analysis of extracellular matrix proteins produced by cultured cells

The extracellular matrix (ECM) is a highly organized multimolecular structure essential for the vital functions of any organism. Although much of the data of extracellular matrix components has been accumulated, the isolation of an entire set of these proteins remains a complex procedure due to the high content of fibrillar proteins and proteoglycans, which form multidomain, netlike structures. In the study presented, we developed a method for isolating ECM proteins from cell cultures. Human epidermoid carcinoma cells A431 and fibroblasts obtained from normal and scar human skin were used. We showed that EDTA solution removed cells from culture plates without destroying the cell membranes. Subsequent treatment of remaining ECM proteins with acetic acid in order to dissociate collagen fibers significantly improved the fractioning of ECM proteins. The extraction of remaining proteins from the surface of the culture plate was preformed by a buffer developed based on Laemmli probe buffer. Using this method, we isolated ECM proteins synthesized by cultured cells, and the extracted proteins were suitable for future analysis by SDS PAGE and two-dimentional electrophoresis, as well as for identifying individual proteins by mass spectrometry. This study may allow us to compare assortments of ECM proteins isolated from different sources, and elucidate impact of various proteins on structure and property of extracellular matrix of investigated cells.     

Assessment of the proliferation of human mesenchymal stromal cells in the presence of human demineralised bone matrix

Tissue engineering integrates discoveries from biochemistry, cell and molecular biology, material science and biomedical engineering to produce innovative three-dimensional composites that can be used to replace or correct damaged tissues and organs. Precise classification of osteoinductive properties of human demineralised bone is often the problem, because it varies from batch to batch. An in vitro assay using bone marrow derived human mesenchymal stromal cells (hMSCs) was developed to improve the classification of the osteoinductive quality of demineralised bone matrix. In this study, three-dimensional, partially demineralised bone scaffolds were investigated for their ability to induce osteogenic differentiation of hMSCs in vitro. Proliferation of the hMSCs was measured by the CelTiter 96? AQueous One Solution Cell assay. Chemical structure was evaluated using quantitative and qualitative X-ray analysis. Scanning electron microscopy revealed primary proliferation of the cells cultivated 14 days and showed elevations in the content of Ca²⁺. These results demonstrate that partially demineralised human bone material supports osteogenic differentiation of hMSCs in vitro. This study documents that in vitro test using hMSCs can be used for classification of the osteoinductive quality of human demineralised bone matrix.     

Non-multipotent stroma inhibit the proliferation and differentiation of mesenchymal stromal cells in vitro

Background aimsThe ability to expand and maintain bone marrow (BM)-derived mesenchymal stem cells (MSC) in vitro is an important aspect of their therapeutic potential. Despite this, the exact composition of stromal cell types within these cultures and the potential effects of non-stem cells on the maintenance of MSC are poorly understood.MethodsC57BL/6J BM stroma was investigated as a model to determine the relationship between MSC and non-multipotent cells in vitro. Whole BM and single-cell derived cultures were characterized using flow cytometry and cell sorting combined with multipotent differentiation. Proliferation of individual stromal populations was evaluated using BrdU.ResultsAt a single-cell level, MSC were distinguished from committed progenitors, and cells lacking differentiation ability, by the expression of CD105 (CD105⁺). A 3-fold reduction in the percentage of CD105⁺ cells was detected after prolonged culture and correlated with loss of MSC. Depletion of CD105⁺ cells coincided with a 10–20% increase in the frequency of proliferating CD105^? cells. Removal of CD105^? stroma caused increased proliferation in CD105⁺ cells, which could be diminished by conditioned media from parent cultures. Comparison of the multipotent differentiation potential in purified and non-purified CD105⁺ cells determined that MSC were detectable for at least 3 weeks longer when cultured in the absence of CD105^? cells.ConclusionsThis work identifies a simple model for characterizing the different cellular components present in BM stromal cultures and demonstrates that stromal cells lacking multipotent differentiating capacity greatly reduce the longevity of MSC.     

Bortezomib enhances the osteogenic differentiation capacity of human mesenchymal stromal cells derived from bone marrow and placental tissues

Bortezomib (BZB) is a chemotherapeutic agent approved for treating multiple myeloma (MM) patients. In addition, there are several reports showing that bortezomib can induce murine mesenchymal stem cells (MSCs) to undergo osteogenic differentiation and increase bone formation in vivo. MSCs are the multipotent stem cells that have capacity to differentiate into several mesodermal derivatives including osteoblasts. Nowadays, MSCs mostly bone marrow derived have been considered as a valuable source of cell for tissue replacement therapy. In this study, the effect of bortezomib on the osteogenic differentiation of human MSCs derived from both bone marrow (BM-MSCs) and postnatal sources such as placenta (PL-MSCs) were investigated. The degree of osteogenic differentiation of BM-MSCs and PL-MSCs after bortezomib treatment was assessed by alkaline phosphatase (ALP) activity, matrix mineralization by Alizarin Red S staining and the expression profiles of osteogenic differentiation marker genes, Osterix, RUNX2 and BSP. The results showed that 1 nM and 2 nM BZB can induce osteogenic differentiation of BM-MSCs and PL-MSCs as demonstrated by increased ALP activity, increased matrix mineralization and up-regulation of osteogenic differentiation marker genes, Osterix, RUNX2 and BSP as compared to controls. The enhancement of osteogenic differentiation of MSCs by bortezomib may lead to the potential therapeutic applications in human diseases especially patients with osteopenia.     

Sulfated glycoproteins and extracellular matrix of cultured human pulmonary endothelial cells

Endothelial cells derived from human pulmonary arteries incorporate (³H)-glucosamine and ³⁵SO₄ into glycosaminoglycans and into the carbohydrate side chains of glycoproteins. These ³H/³⁵S-carbohydrate chains were isolated from cells and culture medium after Pronase digestion. The ³H/³⁵S-glycosaminoglycans were separated from the ³H/³⁵S glycopeptides by chromatography on Sephadex G-50. The distribution of cellular glycosaminoglycans and glycopeptides indicated that 30–60% of the cellular ³⁵S-glycopeptides may be associated with the matrix components that are synthesized by the cell and attached to a plastic substratum. Human pulmonary arterial endothelial cells were grown on collagen or on a matrix derived from vascular smooth muscle cells in order to investigate how smooth muscle cell extracellular matrix components may regulate the synthesis of endothelial cell glycoconjugates. Endothelial cells grown on plastic release various proportions of the glycoconjugates they synthesize into the culture medium. However, these same cells, when grown on substratum composed of extracellular matrix materials, synthesized altered proportions of cell-associated glycosaminoglycans and reduced the levels of total glycosaminoglycans they released into the culture medium. Thus the growth of endothelial cells on a matrix of smooth muscle cell components indicates that the glycosaminoglycan materials released into the culture medium by cells grown on a plastic substratum may not be an accurate reflection of the levels or composition of extracellular matrix materials made by endothelial cells in vivo.     

Decellularized matrix of adipose-derived mesenchymal stromal cells enhanced retinal progenitor cell proliferation via the Akt/Erk pathway and neuronal differentiation

Background aims

Retinal progenitor cells (RPCs) are a promising cell therapy treatment for retinal degenerative diseases. However, problems with limited proliferation ability and differentiation preference toward glia rather than neurons restrict the clinical application of these RPCs. The extracellular matrix (ECM) has been recognized to provide an appropriate microenvironment to support stem cell adhesion and direct cell behaviors, such as self-renewal and differentiation.

Efficient isolation and proliferation of human adipose-derived mesenchymal stromal cells in xeno-free conditions

Molecular Biology Reports - Classical methods used for culture of adipose-derived mesenchymal stromal cells (ADSCs) use xenobiotic components, which may present a potential risk for biological...     

Activated macrophages digest the extracellular matrix proteins produced by cultured cells.

Activated mouse peritoneal macrophages were cultured directly on the extracellular matrix proteins produced by smooth muscle cells $\text{in}\text{vitro}$. The breakdown of the connective tissue proteins to the level of amino acids was followed by observing the release of radioactivity from matrices labelled with [³H]proline. These studies showed that macrophages produce enzymes capable of digesting the matrix and indicated a major role for the macrophage plasminogen activator in this digestion.     

Decreased hypertrophic differentiation accompanies enhanced matrix formation in co-cultures of outer meniscus cells with bone marrow mesenchymal stromal cells

Introduction

The main objective of this study was to determine whether meniscus cells from the outer (MCO) and inner (MCI) regions of the meniscus interact similarly to or differently with mesenchymal stromal stem cells (MSCs). Previous study had shown that co-culture of meniscus cells with bone marrow-derived MSCs result in enhanced matrix formation relative to mono-cultures of meniscus cells and MSCs. However, the study did not examine if cells from the different regions of the meniscus interacted similarly to or differently with MSCs.

Methods

Human menisci were harvested from four patients undergoing total knee replacements. Tissue from the outer and inner regions represented pieces taken from one third and two thirds of the radial distance of the meniscus, respectively. Meniscus cells were released from the menisci after collagenase treatment. Bone marrow MSCs were obtained from the iliac crest of two patients after plastic adherence and in vitro culture until passage 2. Primary meniscus cells from the outer (MCO) or inner (MCI) regions of the meniscus were co-cultured with MSCs in three-dimensional (3D) pellet cultures at 1:3 ratio, respectively, for 3 weeks in the presence of serum-free chondrogenic medium containing TGF-β1. Mono-cultures of MCO, MCI and MSCs served as experimental control groups. The tissue formed after 3 weeks was assessed biochemically, histochemically and by quantitative RT-PCR.

Results

Co-culture of inner (MCI) or outer (MCO) meniscus cells with MSCs resulted in neo-tissue with increased (up to 2.2-fold) proteoglycan (GAG) matrix content relative to tissues formed from mono-cultures of MSCs, MCI and MCO. Co-cultures of MCI or MCO with MSCs produced the same amount of matrix in the tissue formed. However, the expression level of aggrecan was highest in mono-cultures of MSCs but similar in the other four groups. The DNA content of the tissues from co-cultured cells was not statistically different from tissues formed from mono-cultures of MSCs, MCI and MCO. The expression of collagen I (COL1A2) mRNA increased in co-cultured cells relative to mono-cultures of MCO and MCI but not compared to MSC mono-cultures. Collagen II (COL2A1) mRNA expression increased significantly in co-cultures of both MCO and MCI with MSCs compared to their own controls (mono-cultures of MCO and MCI respectively) but only the co-cultures of MCO:MSCs were significantly increased compared to MSC control mono-cultures. Increased collagen II protein expression was visible by collagen II immuno-histochemistry. The mRNA expression level of Sox9 was similar in all pellet cultures. The expression of collagen × (COL10A1) mRNA was 2-fold higher in co-cultures of MCI:MSCs relative to co-cultures of MCO:MSCs. Additionally, other hypertrophic genes, MMP-13 and Indian Hedgehog (IHh), were highly expressed by 4-fold and 18-fold, respectively, in co-cultures of MCI:MSCs relative to co-cultures of MCO:MSCs.

Conclusions

Co-culture of primary MCI or MCO with MSCs resulted in enhanced matrix formation. MCI and MCO increased matrix formation similarly after co-culture with MSCs. However, MCO was more potent than MCI in suppressing hypertrophic differentiation of MSCs. These findings suggest that meniscus cells from the outer-vascular regions of the meniscus can be supplemented with MSCs in order to engineer functional grafts to reconstruct inner-avascular meniscus.     

The immunosuppressive capacity of human mesenchymal stromal cells derived from amnion and bone marrow

Mesenchymal stromal cells derived from amnion (AM-MSCs) can be easily obtained in large quantity by less invasive method in comparison to bone marrow-derived MSCs (BM-MSCs). However, the biological and immunosuppressive properties of AM-MSCs are still poorly characterized. Previous studies demonstrated that BM-MSCs expressed indoleamine 2,3-dioxygenase (IDO) to suppress T-cell responses. This study was designed to address whether IDO contributes to the immunosuppressive function of AM-MSCs. MSCs isolated from amnion were cultured in complete medium similar to BM-MSCs. After culture, AM-MSCs exhibited spindle shape morphology and expressed MSC markers similar to that of BM-MSCs. In addition, AM-MSCs were able to differentiate into adipocytes and osteoblasts. Fascinatingly, AM-MSCs and BM-MSCs exhibited comparable degree of immunosuppressive effect when they were co-cultured with activated T-cells. In addition, IDO secreted by AM-MSCs was responsible for induction of immunosuppressive activities in the same manner as BM-MSCs. Taken together; the results of the present study demonstrate that while AM-MSCs and BM-MSCs show similar immunosuppressive effect, AM-MSCs may have additional advantage over the BM-MSCs in terms of availability. Therefore, AM-MSCs might be considered a potential source for therapeutic applications especially for treatment of immune related diseases.     

Increased proliferation and chemosensitivity of human mesenchymal stromal cells expressing fusion yeast cytosine deaminase

Mesenchymal stromal cells (MSCs) are considered to be suitable vehicles for cellular therapy in various conditions. The expression of reporter and/or effector protein(s) enabled both the identification of MSCs within the organism and the exploitation in targeted tumor therapies. The aim of this study was to evaluate cellular changes induced by retrovirus-mediated transgene expression in MSCs in vitro. Human Adipose Tissue-derived MSCs (AT-MSCs) were transduced to express (i) the enhanced green fluorescent protein (EGFP) reporter transgene, (ii) the fusion yeast cytosine deaminase::uracil phosphoribosyltransferase (CDy::UPRT) enzyme along with the expression of dominant positive selection gene NeoR or (iii) the selection marker NeoR alone (MOCK). CDy::UPRT expression resulted in increased proliferation of CDy::UPRT-MSCs versus naïve AT-MSCs, MOCK-MSCs or EGFP-MSCs. Furthermore, CDy::UPRT-MSCs were significantly more sensitive to 5-fluorouracil (5FU), cisplatin, cyclophosphamide and cytosine arabinoside as determined by increased Caspase 3/7 activation and/or decreased relative proliferation. CDy::UPRT-MSCs in direct cocultures with breast cancer cells MDA-MB-231 increased tumor cell killing induced by low concentrations of 5FU. Our data demonstrated the changes in proliferation and chemoresistance in engineered MSCs expressing transgene with enzymatic function and suggested the possibilities for further augmentation of targeted MSC-mediated antitumor therapy.     

Impaired differentiation potential of human trabecular bone mesenchymal stromal cells from elderly patients

Background aimsAdvances in bone tissue engineering with mesenchymal stromal cells (MSC) as an alternative to conventional orthopedic procedures has opened new horizons for the treatment of large bone defects. Bone marrow (BM) and trabecular bone are both sources of MSC. Regarding clinical use, we tested the potency of MSC from different sources.MethodsWe obtained MSC from 17 donors (mean age 64.6 years) by extensive washing of trabecular bone from the femoral head and trochanter, as well as BM aspirates of the iliac crest and trochanter. The starting material was evaluated by histologic analysis and assessment of colony-forming unit–fibroblasts (CFU-F). The MSC populations were compared for proliferation and differentiation potential, at RNA and morphologic levels.ResultsMSC proliferation potential and immunophenotype (expression of CD49a, CD73, CD90, CD105, CD146 and Stro-1) were similar whatever the starting material. However, the differentiation potential of MSC obtained by bone washing was impaired compared with aspiration; culture-amplified cells showed few Oil Red O-positive adipocytes and few mineralized areas and formed inconsistent Alcian blue-positive high-density micropellets after growth under adipogenic, osteogenic and chondrogenic conditions, respectively. MSC cultured with 1 ng/mL fibroblast growth factor 2 (FGF-2) showed better differentiation potential.ConclusionsTrabecular bone MSC from elderly patients is not good starting material for use in cell therapy for bone repair and regeneration, unless cultured in the presence of FGF-2.     

Enhanced differentiation potential of human amniotic mesenchymal stromal cells by using three-dimensional culturing

The therapeutic potential of human amniotic mesenchymal stromal cells (hAMSCs) remains limited because of their differentiation towards mesenchymal stem cells (MSCs) following adherence. The aim of this study was to develop a three-dimensional (3-D) culture system that would permit hAMSCs to differentiate into cardiomyocyte-like cells. hAMSCs were isolated from human amnions of full-term births collected after Cesarean section. Immunocytochemistry, immunofluorescence and flow cytometry analyses were undertaken to examine hAMSC marker expression for differentiation status after adherence. Membrane currents were determined by patch clamp analysis of hAMSCs grown with or without cardiac lysates. Freshly isolated hAMSCs were positive for human embryonic stem-cell-related markers but their marker profile significantly shifted towards that of MSCs following adherence. hAMSCs cultured in the 3-D culture system in the presence of cardiac lysate expressed cardiomyocyte-specific markers, in contrast to those maintained in standard adherent cultures or those in 3-D cultures without cardiac lysate. hAMSCs cultured in 3-D with cardiac lysate displayed a cardiomyocyte-like phenotype as observed by membrane currents, including a calcium-activated potassium current, a delayed rectifier potassium current and a Ca²⁺-resistant transient outward K⁺ current. Thus, although adherence limits the potential of hAMSCs to differentiate into cardiomyocyte-like cells, the 3-D culture of hAMSCs represents a more effective method of their culture for use in regenerative medicine.