Enhancing survival,engraftment, and osteogenic potential of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are promising candidates for bone regeneration therapies due to their plasticity and easiness of sourcing. MSC-based treatments are generally considered a safe procedure, however, the long-term results obtained up to now are far from satisfactory. The main causes of these therapeutic limitations are inefficient homing, engraftment, and osteogenic differentiation. Many studies have proposed modifications to improve MSC engraftment and osteogenic differentiation of the transplanted cells. Several strategies are aimed to improve cell resistance to the hostile microenvironment found in the recipient tissue and increase cell survival after transplantation. These strategies could range from a simple modification of the culture conditions, known as cell-preconditioning, to the genetic modification of the cells to avoid cellular senescence. Many efforts have also been done in order to enhance the osteogenic potential of the transplanted cells and induce bone formation, mainly by the use of bioactive or biomimetic scaffolds, although alternative approaches will also be discussed. This review aims to summarize several of the most recent approaches, providing an up-to-date view of the main developments in MSC-based regenerative techniques.     

Human mesenchymal stem cells express neuronal markers after osteogenic and adipogenic differentiation

Mesenchymal stem cells (MSCs) are multipotent cells that are able to differentiate into mesodermal lineages (osteogenic, adipogenic, chondrogenic), but also towards non-mesodermal derivatives (e.g. neural cells). Recent in vitro studies revealed that, in the absence of any kind of differentiation stimuli, undifferentiated MSCs express neural differentiation markers, but the literature data do not all concur. Considering their promising therapeutic potential for neurodegenerative diseases, it is very important to expand our knowledge about this particular biological property of MSCs. In this study, we confirmed the spontaneous expression of neural markers (neuronal, glial and progenitor markers) by undifferentiated human MSCs (hMSCs) and in particular, we demonstrated that the neuronal markers βIII-tubulin and NeuN are expressed by a very high percentage of hMSCs, regardless of the number of culture passages and the culture conditions. Moreover, the neuronal markers βIII-tubulin and NeuN are still expressed by hMSCs after in vitro osteogenic and adipogenic differentiation. On the other hand, chondrogenically differentiated hMSCs are negative for these markers. Our findings suggest that the expression of neuronal markers could be common to a wide range of cellular types and not exclusive for neuronal lineages. Therefore, the expression of neuronal markers alone is not sufficient to demonstrate the differentiation of MSCs towards the neuronal phenotype. Functional properties analysis is also required.     

Influence of obstetric factors on osteogenic potential of umbilical cord-derived mesenchymal stem cells

Wharton's jelly from the umbilical cord is a noncontroversial source of mesenchymal stem cells (WJMSCs) with high plasticity, proliferation rate and ability to differentiate towards multiple lineages. WJMSCs from different donors have been characterized for their osteogenic potential. Although there is large evidence of WJMSCs plasticity, recently scientific debate has focused on MSCs selection, establishing predictable elements to discriminate the cells with most promising osteoprogenitor cell potential.     

Fates and osteogenic differentiation potential of human mesenchymal stem cells in immunocompromised mice

Human mesenchymal stem cells (hMSCs) from bone marrow were genetically marked by using a murine leukaemia virus construct encoding enhanced green fluorescent protein (eGFP). The marked cells were either directly implanted into the tibialis anterior muscle or introduced into a variety of other tissue sites in immunocompromised mice (NOD/SCID and C.B-17 SCID/beige) to investigate their fates and differentiation potentials. It was observed that the hMSCs survived for up to 12 weeks and showed site-specific morphological phenotypes. hMSCs delivered by intravenous injection were found mainly in the lungs and were detected rarely in other organs. Histomorphometry showed that, after implantation of hMSCs into the tibialis anterior muscle juxtaskeletally, the areas of reactive host callus formation at 1 and 2 weeks and of ectopic human bone formation at 1 week were significantly increased compared with the control group. Expression of eGFP and human RUNX2, alkaline phosphatase, osteocalcin, osteopontin, and collagen type I mRNAs were detected in mice implanted with the labelled hMSCs but not in sham-treated samples. Active clearance of the reactive callus and ectopic calcified tissue by osteoclast-like tartrate-resistant acid phosphatase-positive cells was observed. We conclude that the eGFP-labelled hMSCs can survive and retain the potential to differentiate morphologically into a variety of apparent mesenchymal phenotypes in vivo. Absolute confirmation of differentiation capacity requires further study and is complicated by known possibilities of fusion of donor and host cells or limited transfer of genetic material. Nevertheless, the genetically marked hMSCs are shown to participate extensively in bone formation and turnover. Control of the host osteoclast/macrophage responses resulting in clearance of formed osteogenic tissue warrants further investigation to promote prolonged human osteogenesis in immunocompromised mice. Furthermore, any proposed general cytotherapeutic strategy for enhanced osteogenesis is likely to require supplementation of local bone-forming biological signals.     

In vitro augmentation of mesenchymal stem cells viability in stressful microenvironments: In vitro augmentation of mesenchymal stem cells viability

Mesenchymal stem cells (MSCs) are under intensive investigation for use in cell-based therapies because their differentiation abilities, immunomodulatory effects, and homing properties offer potential for significantly augmenting regenerative capacity of many tissues. Nevertheless, major impediments to their therapeutic application, such as low proliferation and survival rates remain as obstacles to broad clinical use of MSCs. Another major challenge to evolution of MSC-based therapies is functional degradation of these cells as a result of their exposure to oxidative stressors during isolation. Indeed, oxidative stress-mediated MSC depletion occurs due to inflammatory processes associated with chemotherapy, radiotherapy, and expression of pro-apoptotic factors, and the microenvironment of damaged tissue in patients receiving MSC therapy is typically therapeutic not favorable to their survival. For this reason, any strategies that enhance the viability and proliferative capacity of MSCs associated with their therapeutic use are of great value. Here, recent strategies used by various researchers to improve MSC allograft function are reviewed, with particular focus on in vitro conditioning of MSCs in preparation for clinical application. Preconditioning, genetic manipulation, and optimization of MSC culture conditions are some examples of the methodologies described in the present article, along with novel strategies such as treatment of MSCs with secretome and MSC-derived microvesicles. This topic material is likely to find value as a guide for both research and clinical use of MSC allografts and for improvement of the value that use of these cells brings to health care.     

Molybdenum trioxide enhances viability,osteogenic differentiation and extracellular matrix formation of human bone marrow-derived mesenchymal stromal cells

BackgroundMetals and their ions allow specific modifications of the biological properties of bioactive materials that are intended for application in bone tissue engineering. While there is some evidence about the impact of particles derived from orthopedic Cobalt-Chromium-Molybdenum (Co-Cr-Mo) alloys on cells, there is only limited data regarding the influence of the essential trace element Mo and its ions on the viability, osteogenic differentiation as well as on the formation and maturation of the primitive extracellular matrix (ECM) of primary human bone marrow-derived stromal cells (BMSCs) available so far.MethodsIn this study, the influence of a wide range of molybdenum (VI) trioxide (MoO₃), concentrations on BMSC viability was evaluated via measurement of fluorescein diacetate metabolization. Thereafter, the impact of three non-cytotoxic concentrations of MoO₃ on the cellular osteogenic differentiation as well as on ECM formation and maturation of BMSCs was assessed.ResultsMoO₃ had no negative influence on BMSC viability in most tested concentrations, as viability was in fact even enhanced. Only the highest concentration (10 mM) of MoO₃ showed cytotoxic effects. Cellular osteogenic differentiation, measured via the marker enzyme alkaline phosphatase was enhanced by the presence of MoO₃ in a concentration-dependent manner. Furthermore, MoO₃ showed a positive influence on the expression of relevant marker genes for osteogenic differentiation (osteopontin, osteocalcin and type I collagen alpha 1) and on the formation and maturation of the primitive ECM, as measured by collagen deposition and ECM calcification.ConclusionMoO₃ is considered as an attractive candidate for supplementation in biomaterials and qualifies for further research.     

Laminin-5 activates extracellular matrix production and osteogenic gene focusing in human mesenchymal stem cells.

We recently reported that laminin-5, expressed by human mesenchymal stem cells (hMSC), stimulates osteogenic gene expression in these cells in the absence of any other osteogenic stimulus. Here we employ two-dimensional liquid chromatography and tandem mass spectrometry, along with the Database for Annotation, Visualization and Integrated Discovery (DAVID), to obtain a more comprehensive profile of the protein (and hence gene) expression changes occurring during laminin-5-induced osteogenesis of hMSC. Specifically, we compare the protein expression profiles of undifferentiated hMSC, hMSC cultured on laminin-5 (Ln-5 hMSC), and fully differentiated human osteoblasts (hOST) with profiles from hMSC treated with well-established osteogenic stimuli (collagen I, vitronectin, or dexamethazone). We find a marked reduction in the number of proteins (e.g., those involved with calcium signaling and cellular metabolism) expressed in Ln-5 hMSC compared to hMSC, consistent with our previous finding that hOST express far fewer proteins than do their hMSC progenitors, a pattern we call "osteogenic gene focusing." This focused set, which resembles an intermediate stage between hMSC and mature hOST, mirrors the expression profiles of hMSC exposed to established osteogenic stimuli and includes osteogenic extracellular matrix proteins (collagen, vitronectin) and their integrin receptors, calcium signaling proteins, and enzymes involved in lipid metabolism. These results provide direct evidence that laminin-5 alone stimulates global changes in gene/protein expression in hMSC that lead to commitment of these cells to the osteogenic phenotype, and that this commitment correlates with extracellular matrix production.     

Human fetal mesenchymal stem cells

Stem cells have been isolated at all stages of development from the early developing embryo to the post-reproductive adult organism. However, the fetal environment is unique as it is the only time in ontogeny that there is migration of stem cells in large numbers into different organ compartments. While fetal neural and haemopoietic stem cells (HSC) have been well characterised, only recently have mesenchymal stem cells from the human fetus been isolated and evaluated. Our group have characterised in human fetal blood, liver and bone marrow a population of non-haemopoietic, non-endothelial cells with an immunophenotype similar to adult bone marrow-derived mesenchymal stem cells (MSC). These cells, human fetal mesenchymal stem cells (hfMSC), are true multipotent stem cells with greater self-renewal and differentiation capacity than their adult counterparts. They circulate in first trimester fetal blood and have been found to traffic into the maternal circulation, engrafting in bone marrow, where they remain microchimeric for decades after pregnancy. Though fetal microchimerism has been implicated in the pathogenesis of autoimmune disease, the biological role of hfMSC microchimerism is unknown. Potential downstream applications of hfMSC include their use as a target cell for non-invasive pre-natal diagnosis from maternal blood, and for fetal cellular and gene therapy. Using hfMSC in fetal therapy offers the theoretical advantages of avoidance of immune rejection, increased engraftment, and treatment before disease pathology sets in. Aside from allogeneic hfMSC in utero transplantation, the use of autologous hfMSC has been brought a step forward with the development of early blood sampling techniques, efficient viral transduction and clonal expansion. Work is ongoing to determine hfMSC fate post-transplantation in murine models of genetic disease. In this review we will examine what is known about hfMSC biology, as well as discussing areas for future research. The implications of hfMSC trafficking in pregnancy will be explored and the potential clinical applications of hfMSC in prenatal diagnosis and fetal therapy discussed.     

Nanocomposite scaffold seeded with mesenchymal stem cells for bone repair

The mechanical property of bone tissue scaffolds is one of the most important aspects in bone tissue engineering that has remained problematic. In our previous study, we fabricated a three‐dimensional scaffold from nano‐hydroxyapatite/gelatin (nHA/Gel) and investigated its efficiency in promoting bone regeneration both in vitro and in vivo. In the present study, the effect of adding silicon carbide (SiC) on the mechanical and biological behaviors of the nHA/Gel/SiC and bone regeneration in vivo were determined. nHA and SiC were synthesized and characterized by the X‐ray diffraction pattern and transmission electron microscope image. Layer solvent casting, freeze drying, and lamination techniques were applied to prepare these scaffolds. Then, the biocompatibility and cell adhesion behavior of the synthesized nHA/Gel/SiC scaffolds were investigated. For in vivo studies, rats were categorized into three groups: blank defect, blank scaffold, and rat bone marrow mesenchymal stem cells (rBM‐MSCs)/scaffold. After 1, 4, and 12 weeks post‐injury, the rats were sacrificed and the calvaria were harvested. Sections with a thickness of 5 µm thickness were prepared and stained with hematoxylin–eosin and Masson's Trichrome, and immunohistochemistry was performed. Our results showed that SiC effectively increased the mechanical properties of the nHA/Gel/SiC scaffold. No significant differences were observed in biocompatibility, cell adhesion, and cytotoxicity of the nHA/Gel/SiC in comparison with the nHA/Gel nanocomposite. Based on histological and immunohistochemical studies, both osteogenesis and collagenization were significantly higher in the rBM‐MSCs/scaffold group, quantitatively and qualitatively. The present study strongly suggests the potential of SiC as an alternative strategy to improve the mechanical and biological properties of bone tissue engineering scaffolds, and shows that the pre‐seeded nHA/Gel/SiC scaffold with rBM‐MSCs improves osteogenesis in the engineered bone implant.     

Viability of human mesenchymal stem cells seeded on crosslinked entropy-elastic gelatin-based hydrogels

Biomimetic polymer network systems with tailorable properties based on biopolymers represent a class of degradable hydrogels that provides sequences for protein adsorption and cell adhesion. Such materials show potential for in vitro MSC proliferation as well as in vivo applications and were obtained by crosslinking different concentrations of gelatin using varying amounts of ethyl lysine diisocyanate in the presence of a surfactant in pH 7.4 PBS solution. Material extracts, which were tested for cytotoxic effects using L929 mouse fibroblasts, were non-toxic. The hydrogels were seeded with human bone marrow-derived MSCs and supported viable MSCs for the incubation time of 9 d. Preadsorption of fibronectin on materials improved this biofunctionality.     

Comparing the osteogenic potential of schneiderian membrane and dental pulp mesenchymal stem cells: an in vitro study

Cell and Tissue Banking - Mesenchymal stem cells, being characterized by high self-renewal capacity and multi-lineage differentiation potential, are widely used in regenerative medicine especially...     

Human amnion-derived mesenchymal stem cells are a potential source for uterine stem cell therapy

Abstract. Objectives: Human amnion is an easy‐to‐obtain novel source of human mesenchymal stem cells, which poses little or no ethical dilemmas. We have previously shown that human amnion‐derived mesenchymal (HAM) cells exhibit certain mesenchymal stem cell‐like characteristics with respect to expression of stem cell markers and differentiation potentials. Materials and methods: In this study, we further characterized HAM cells’ potential for in vivo therapeutic application. Results: Flow cytometric analyses of HAM cells show that they express several stem cell‐related cell surface markers, including CD90, CD105, CD59, CD49d, CD44 and HLA‐ABC, but not CD45, CD34, CD31, CD106 or HLA‐DR. HAM cells at the 10th passage showed normal karyotype. More interestingly, the AbdB‐like HOXA genes HOXA9, HOXA10 and HOXA11 that are expressed in the mesenchyme of the developing female reproductive tract and pregnant uteri are also expressed in HAM cells, suggesting similarities between these two mesenchymal cell types. Progesterone receptor is also highly expressed in HAM cells and expression of genes or proteins in HAM cells could be manipulated with the aid of lentivirus technology or cell‐permeable peptides. To test potentials of HAM cells for in vivo application, we introduced enhanced green fluorescence protein (EGFP)‐expressing HAM cells to mice by intrauterine infusion (into uteri) or by intravenous injection (into the circulation). Presence of EGFP‐expressing cells within the uterine mesenchyme after intrauterine infusion or in lungs after intravenous injection was noted within 1–4 weeks. Conclusions: Collectively, these results suggest that HAM cells are a potential source of mesenchymal stem cells with therapeutic potential.     

Self-assembled extracellular macromolecular matrices and their different osteogenic potential with preosteoblasts and rat bone marrow mesenchymal stromal cells

Extracellular environment is a physical support that is critical to cell adhesion, migration, and differentiation. In this work, cell-derived matrices (CDMs) were obtained by separately culturing fibroblasts, preosteoblasts, and chondrocytes. The cells were grown on a coverslip and subjected to decellularization using detergents and enzymes. The resulting matrices were named fibroblast-derived matrix (FDM), preosteoblast-derived matrix (PDM), and chondrocyte-derived matrix (CHDM). We hypothesize that the unique compositional and structural feature of each CDM provides cells with a distinct microenvironment capable of functioning as a different signaling cue in the regulation of preosteoblast and rat bone marrow mesenchymal stromal cell (BMSC) osteogenic differentiation. SEM images show that each cell type creates its unique surface texture in a fibrillar structure. Three major macromolecules, fibronectin, type I collagen, and laminin, were clearly identified using both immunofluorescence and Western blot, in which FDM exhibited a much stronger signal of each ECM component than that of PDM or CHDM. For early cell morphology, BMSCs on the CDMs were highly elongated in a spindle-like shape. Both preosteoblasts and BMSCs proliferated well on CDMs comparable to the control. Once preosteoblasts were cultured for 2 weeks, their osteogenic activity was significantly different depending on the type of CDM. Using Alizarin red and von Kossa staining, we found that the cells on the FDM were much more osteogenic than the other groups. Furthermore, FDM was the most effective in upregulating the osteogenic markers, such as alkaline phosphatase (ALP), osteopontin, osteocalcin, and type I collagen. In particular, we observed a 2.5-fold increase in ALP activity with FDM compared to that of control and CHDM. In stark contrast, CHDM was very poor in stimulating osteogenic differentiation of preosteoblasts. Interestingly, these results were reproducible with the use of BMSCs, which are much more heterogeneous in cell populations than preosteoblasts. CHDM was still very weak in triggering the osteogenesis of BMSCs, whereas both FDM and PDM were equally competitive. This study demonstrates that a combination of factors (surface texture and composition) shape a unique cellular microenvironment, which serves as a physical cue toward the osteogenic differentiation of preosteoblasts and BMSCs.     

Calcium phosphate surfaces promote osteogenic differentiation of mesenchymal stem cells

Although studies in vivo revealed promising results in bone regeneration after implantation of scaffolds together with osteogenic progenitor cells, basic questions remain how material surfaces control the biology of mesenchymal stem cells (MSC). We used human MSC derived from bone marrow and studied the osteogenic differentiation on calcium phosphate surfaces. In osteogenic differentiation medium MSC differentiated to osteoblasts on hydroxyapatite and BONITmatrix, a degradable xerogel composite, within 14 days. Cells revealed a higher alkaline phosphatase (ALP) activity and increased RNA expression of collagen I and osteocalcin using real-time RTPCR compared with cells on tissue culture plastic. To test whether material surface characteristics alone are able to stimulate osteogenic differentiation, MSC were cultured on the materials in expansion medium without soluble additives for osteogenic differentiation. Indeed, cells on calcium phosphate without osteogenic differentiation additives developed to osteoblasts as shown by increased ALP activity and expression of osteogenic genes, which was not the case on tissue culture plastic. Because we reasoned that the stimulating effect on osteogenesis by calcium phosphate surfaces depends on an altered cell-extracellular matrix interaction we studied the dynamic behaviour of focal adhesions using cells transfected with GFP labelled vinculin. On BONITmatrix, an increased mobility of focal adhesions was observed compared with cells on tissue culture plastic. In conclusion, calcium phosphate surfaces are able to drive MSC to osteoblasts in the absence of osteogenic differentiation supplements in the medium. An altered dynamic behaviour of focal adhesions on calcium phosphate surfaces might be involved in the molecular mechanisms which promote osteogenic differentiation.     

Ovine bone marrow mesenchymal stem cells: isolation and characterization of the cells and their osteogenic differentiation potential on embroidered and surface-modified polycaprolactone-co-lactide scaffolds

The current study was undertaken with the goal being isolation, cultivation, and characterization of ovine mesenchymal stem cells (oMSC). Furthermore, the objective was to determine whether biological active polycaprolactone-co-lactide (trade name PCL) scaffolds support the growth and differentiation of oMSC in vitro. The oMSC were isolated from the iliac crest of six merino sheep. Three factors were used to demonstrate the MSC properties of the isolated cells in detail. (1) Their ability to proliferate in culture with a spindle-shaped morphology, (2) presence of specific surface marker proteins, and (3) their capacity to differentiate into the three classical mesenchymal pathways, osteoblastic, adipogenic, and chondrogenic lineages. Furthermore, embroidered PCL scaffolds were coated with collagen I (coll I) and chondroitin sulfate (CS). The porous structure of the scaffolds and the coating with coll I/CS allowed the oMSC to adhere, proliferate, and to migrate into the scaffolds. The coll I/CS coating on the PCL scaffolds induced osteogenic differentiation of hMSC, without differentiation supplements, indicating that the scaffold also has an osteoinductive character. In conclusion, the isolated cells from the ovine bone marrow have similar morphologic, immunophenotypic, and functional characteristics as their human counterparts. These cells were also found to differentiate into multiple mesenchymal cell types. This study demonstrates that embroidered PCL scaffolds can act as a temporary matrix for cell migration, proliferation, and differentiation of oMSC. The data presented will provide a reliable model system to assess the translation of MSC-based therapy into a variety of valuable ovine experimental models under autologous settings.