Overview of mesenchymal stem cells

Stem cells are unprogrammed cells which possess plasticity and self renewal capability. The term of stem cell was first used to describe cells committed to give rise to germline cells, and to describe proposed progenitor cells of the blood system [1]. A unique feature of stem cell is to remain quiescent in vivo in an uncommitted state. They serve as reservoir or natural support system to replenish cells lost due to disease, injury or aging. When triggered by appropriate signals these cells divide and may become specialized, committed cells; however being able to control this differentiation process still remains one of the biggest challenge in stem cell research [2]. The cell division of stem cells is a distinct aspect of their biology, since this division may be either symmetric or asymmetric. Symmetric division takes place when the stem cells divides and forms two new daughter cells. Asymmetric division is thought to take place only under certain conditions where stem cells divides and gives rise to a daughter cell which remains primitive and does not proliferate, and one committed progenitor cell, which heads down a path of differentiation. Asymmetric division of stem cells helps reparative process, and also ensures that the stem cells pool does not decrease, whereas symmetric division is responsible for stem cells undergoing self renewal and proliferation. The factors which prompt the stem cells to undergo asymmetric division are, however, not well understood, but it is clear that the delicate balance between the self renewal and differentiation is what maintains tissue homeostasis.

Mesenchymal stem cells in osteoarthritis therapy : current status of theory, technology, and applications

Osteoarthritis (OA) is a chronic, non-inflammatory type of arthritis, which usually affects the movable and weight bearing joints of the body. It is the most common joint disease in human beings and common in elderly people. Till date, there are no safe and effective diseases modifying OA drugs (DMOADs) to treat the millions of patients suffering from this serious and debilitating disease. However, recent studies provide strong evidence for the use of mesenchymal stem cell (MSC) therapy in curing cartilage related disorders. Due to their natural differentiation properties, MSCs can serve as vehicles for the delivery of effective, targeted treatment to damaged cartilage in OA disease. In vitro, MSCs can readily be tailored with transgenes with anti-catabolic or pro-anabolic effects to create cartilage-friendly therapeutic vehicles. On the other hand, tissue engineering constructs with scaffolds and biomaterials holds promising biological cartilage therapy. Many of these strategies have been validated in a wide range of in vitro and in vivo studies assessing treatment feasibility or efficacy. In this review, we provide an outline of the rationale and status of stem-cell-based treatments for OA cartilage, and we discuss prospects for clinical implementation and the factors crucial for maintaining the drive towards this goal.

Application of hyperbaric oxygen in bone tissue engineering : effect of hyperbaric oxygen treatment on bone marrow stem cells

and non-union of bony fractures has been proposed since 1966, little has been known about the effect of HBOT on bone marrow stem cells (BMSC). The aim of this study is to investigate the effect of HBO treatment on osteogenetic differentiation of BMSC and potential application in bone tissue engineering. Adhesive stromal cells harvested from bone marrow were characterized by mesenchymal differentiation potential, cell surface markers and their proliferation capacity. Mesenchymal stem cells, which demonstrated osteogenic, chondrogenic and adipogenic differentiation potential and expressed positively for CD 29, CD 44, CD 73, CD 90, CD 105, CD 166 and negatively for CD34 and CD 45, were selected and treated in a laboratory-scale HBO chamber using different oxygen pressures and exposure times. No obvious effect of HBO treatment on BMSC proliferation was noticed. However, cytotoxic effects of HBO were considerably less pronounced when cells were cultured in medium supplemented with 10% FBS in comparison to medium supplemented with 2% FCS, as was evaluated by WST-1 assay. Under HBO treatment, bone nodules were formed in three days, which was clearly revealed by Von Kossa staining. In contrasts, without HBO treatment, bone nodules were not detected until 9-12 days using the same inducing culture media. Calcium deposition was also significantly increased after three days of HBO treatments compared to no HBO treatment. In addition it was also found that oxygen played a direct role in the enhancement of BMSC osteogenic differentiation, which was independent of the effect of air pressure.

Strategy in developing cell based therapy for large bone

Regeneration of osseous defects by tissue-engineering approach provides a novel means of treatment utilizing cell biology, materials science, and molecular biology. The concept of in vitro cultured osteoblasts having an ability to induce new bone formation has been demonstrated in the critical size defects using small animal models. The bone derived cells can be incorporated into bioengineered scaffolds and synthesize bone matrix, which on implantation can induce new bone formation. In search of optimal cell delivery materials, the extracellular matrix as cell carriers for the repair and regeneration of tissues is receiving increased attention. We have investigated extracellular matrix formed by osteoblasts in vitro as a scaffold for osteoblasts transplantation and found a mineralized matrix, formed by human osteoblasts in vitro, can initiate bone formation by activating endogenous mesenchymal cells. To repair the large bone defects, osteogenic or stem cells need to be prefabricated in a large three dimensional scaffold usually made of synthetic biomaterials, which have inadequate interaction with cells and lead to in vivo foreign body reactions. The interstitial extracellular matrix has been applied to modify biomaterials surface and identified vitronectin, which binds the heparin domain and RGD (Arg-Gly-Asp) sequence can modulate cell spreading, migration and matrix formation on biomaterials. We also synthesized a tri-block copolymer, methoxy-terminated poly(ethylene glycol)(MPEG)-polyL-lactide(PLLA)-polylysine(PLL) for human osteoblasts delivery. We identified osteogenic activity can be regulated by the molecular weight and composition of the triblock copolymers. Due to the sequential loss of lineage differentiation potential during the culture of bone marrow stromal cells that hinderers their potential clinical application, we have developed a clonal culture system and established several stem cell clones with fast growing and multi-differentiation properties. Using proteomics and subtractive immunization, several differential proteins have been identified and verified their potential application in stem cell characterization and tissue regeneration

The effects of pore architecture in silk fibroin scaffolds on the growth and differentiation of BMP7-expressing mesenchymal stem cells

Pore architecture of scaffolds is known to play a critical role in tissue engineering as it provides the vital framework for the seeded cells to organize into a functioning tissue. In this report, we investigated the effects of different concentration on silk fibroin protein 3D scaffold pore microstructure. Four pore size ranges of silk fibroin scaffolds were made by freeze-dry technique, with the pore sizes ranging from 50 to 300 µm. The pore size of the scaffold decreases as the concentration increases. Human mesenchymal stem cells were in vitro cultured in these scaffolds. After BMP7 gene transferred, DNA assay, ALP assay, hematoxylin–eosin staining, alizarin red staining and reverse transcription-polymerase chain reaction were performed to analyze the effect of the pore size on cell growth, differentiation and the secretion of extracellular matrix (ECM). Cell morphology in these 3D scaffolds was investigated by confocal microscopy. This study indicates mesenchymal stem cells prefer the group of scaffolds with pore size between 100 and 300 µm for better proliferation and ECM production

The ratio of VEGF/PEDF expression in bone marrow mesenchymal stem cells regulates neovascularization

Angiogenesis, or neovascularization, is a finely balanced process controlled by pro- and anti-angiogenic factors. Vascular endothelial growth factor (VEGF) is a major pro-angiogenic factor, whereas pigment epithelial-derived factor (PEDF) is the most potent natural angiogenesis inhibitor. In this study, the regulatory role of bone marrow stromal cells (BMSCs) during angiogenesis was assessed by the endothelial differentiation potential, VEGF/PEDF production and responses to pro-angiogenic and hypoxic conditions. The in vivo regulation of blood vessel formation by BMSCs was also explored in a SCID mouse model. Results showed that PEDF was expressed more prominently in BMSCs compared to VEGF. This contrasted with human umbilical vein endothelial cells (HUVECs) where the expression of VEGF was higher than that of PEDF. The ratio of VEGF/PEDF gene expression in BMSCs increased when VEGF concentration reached 40 ng/ml in the culture medium, but decreased at 80 ng/ml. Under CoCl2- induced hypoxic conditions, the VEGF/PEDF ratio of BMSCs increased significantly in both normal and angiogenic culture media. There was no expression of endothelial cell markers in BMSCs cultured in either pro-angiogenic or hypoxia culture conditions when compared with HUVECs. The in vivo study showed that VEGF/PEDF expression closely correlated with the degree of neovascularization, and that hypoxia significantly induced pro-angiogenic activity in BMSCs. These results indicate that, rather than being progenitors of endothelial cells, BMSCs play an important role in regulating the neovascularization process, and that the ratio of VEGF and PEDF may, in effect, be an indicator of the pro- or antiangiogenic activities of BMSCs.

Periodontal regeneration and mesenchymal stem cells

The ultimate goal of periodontal therapy is to regenerate periodontal supporting tissues, but this is hard to achieve as the results of periodontal techniques for regeneration are clinically unpredictable. Stem cells owing to their plasticity and proliferation potential provides a new paradigm for periodontal regeneration. Stem cells from mesenchyme can self renew and generate new dental tissues (including dentin and cementum), alveolar bone and periodontal ligament, and thus they have great potential in periodontal regeneration. This chapter presents an insight into mesenchymal stem cells and their potential use in periodontal regeneration. In this chapter the cellular and molecular biology in periodontal regeneration will be introduced, followed by a range of conventional surgical procedures for periodontal regeneration will be discussed. Mesenchymal stem cells applied in regenerated periodontal tissue and their biological characterizations in vitro will be also introduced. Lastly, the use of mesenchymal stem cell to repair periodontal tissues in large animal models will be also reviewed.

ATF5, a possible regulator of osteogenic differentiation in human adipose-derived stem cells

The regulatory pathways involved in maintaining the pluripotency of embryonic stem cells are partially known, whereas the regulatory pathways governing adult stem cells and their "stem-ness" are characterized to an even lesser extent. We, therefore, screened the transcriptome profiles of 20 osteogenically induced adult human adipose-derived stem cell (ADSC) populations and investigated for putative transcription factors that could regulate the osteogenic differentiation of these ADSC. We studied a subgroup of donors' samples that had a disparate osteogenic response transcriptome from that of induced human fetal osteoblasts and the rest of the induced human ADSC samples. From our statistical analysis, we found activating transcription factor 5 (ATF5) to be significantly and consistently down-regulated in a randomized time-course study of osteogenically differentiated adipose-derived stem cells from human donor samples. Knockdown of ATF5 with siRNA showed an increased sensitivity to osteogenic induction. This evidence suggests a role for ATF5 in the regulation of osteogenic differentiation in adipose-derived stem cells. To our knowledge, this is the first report that indicates a novel role of transcription factors in regulating osteogenic differentiation in adult or tissue specific stem cells. © 2012 Wiley Periodicals, Inc.

Bioreactor for blood product production

The feasibility of ex vivo blood production is limited by both biological and engineering challenges. From an engineering perspective, these challenges include the significant volumes required to generate even a single unit of a blood product, as well as the correspondingly high protein consumption required for such large volume cultures. Membrane bioreactors, such as hollow fiber bioreactors (HFBRs), enable cell densities approximately 100-fold greater than traditional culture systems and therefore may enable a significant reduction in culture working volumes. As cultured cells, and larger molecules, are retained within a fraction of the system volume, via a semipermeable membrane it may be possible to reduce protein consumption by limiting supplementation to only this fraction. Typically, HFBRs are complex perfusion systems having total volumes incompatible with bench scale screening and optimization of stem cell-based cultures. In this article we describe the use of a simplified HFBR system to assess the feasibility of this technology to produce blood products from umbilical cord blood-derived CD34+ hematopoietic stem progenitor cells (HSPCs). Unlike conventional HFBR systems used for protein manufacture, where cells are cultured in the extracapillary space, we have cultured cells in the intracapillary space, which is likely more compatible with the large-scale production of blood cell suspension cultures. Using this platform we direct HSPCs down the myeloid lineage, while targeting a 100-fold increase in cell density and the use of protein-free bulk medium. Our results demonstrate the potential of this system to deliver high cell densities, even in the absence of protein supplementation of the bulk medium.

Organotypic culture of normal, dysplastic and squamous cell carcinoma-derived oral cell lines reveals loss of spatial regulation of CD44 and p75(NTR) in malignancy

Oral squamous cell carcinomas (OSCC) often arise from dysplastic lesions. The role of cancer stem cells in tumour initiation is widely accepted, yet the potential existence of pre-cancerous stem cells in dysplastic tissue has received little attention. Cell lines from oral diseases ranging in severity from dysplasia to malignancy provide opportunity to investigate the involvement of stem cells in malignant progression from dysplasia. Stem cells are functionally defined by their ability to generate hierarchical tissue structures in consortium with spatial regulation. Organotypic cultures readily display tissue hierarchy in vitro; hence, in this study, we compared hierarchical expression of stem cell-associated markers in dermis-based organotypic cultures of oral epithelial cells from normal tissue (OKF6-TERT2), mild dysplasia (DOK), severe dysplasia (POE-9n) and OSCC (PE/CA P J15). Expression of CD44, p75NTR, CD24 and ALDH was studied in monolayers by flow cytometry and in organotypic cultures by immunohistochemistry. Spatial regulation of CD44 and p75NTR was evident for organotypic cultures of normal (OKF6-TERT2) and dysplasia (DOK and POE-9n) but was lacking for OSCC (PE/CA PJ15)-derived cells. Spatial regulation of CD24 was not evident. All monolayer cultures exhibited CD44, p75NTR, CD24 antigens and ALDH activity (ALDEFLUOR® assay), with a trend towards loss of population heterogeneity that mirrored disease severity. In monolayer, increased FOXA1 and decreased FOXA2 expression correlated with disease severity, but OCT3/4, Sox2 and NANOG did not. We conclude that dermis-based organotypic cultures give opportunity to investigate the mechanisms that underlie loss of spatial regulation of stem cell markers seen with OSCC-derived cells.

The key regulatory roles of the PI3K/Akt signalling pathway in the functionalities of mesenchymal stem cells and applications in tissue regeneration

Mesenchymal stem cells (MSCs) are multi-potent cells that can differentiate into various cell types and have been used widely in tissue engineering application. In tissue engineering, a scaffold, MSCs and growth factors are used as essential components and their interactions have been regarded to be important for regeneration of tissues. A critical problem for MSCs in tissue engineering is their low survival ability and functionality. Most MSCs are going to be apoptotic after transplantation. Therefore, increasing MSC survival ability and functionalities is the key for potential applications of MSCs. Several approaches have been studied to increase MSC tissue forming capacity including application of growth factors, overexpression of stem cell regulatory genes and improvement of biomaterials for scaffolds. The effects of these approaches on MSCs have been associated with the activation of the PI3K/Akt signaling pathway. The pathway plays central regulatory roles in MSC survival, proliferation, migration, angiogenesis, cytokine production and differentiation. In this review, we summarize and discuss the literatures related to the roles of the PI3K/Akt pathway in the functionalities of MSCs and the involvement of the pathway in biomaterials-increased MSC functinalities. Biomaterials have been modified in their properties, surface structure and loaded with growth factors to increase MSC functionalities. Several studies demonstrated that the biomaterials-increased MSC functionalities are mediated by the activation of the PI3K/Akt pathway.

Mesenchymal stem cells and nano-structured surfaces

Mesenchymal stem cells (MSCs) represent multipotent stromal cells that can differentiate into a variety of cell types, including osteoblasts (bone cells), chondrocytes (cartilage cells), and adipocytes (fat cells). Their multi-potency provides a great promise as a cell source for tissue engineering and cell-based therapy for many diseases, particularly bone diseases and bone formation. To be able to direct and modulate the differentiation of MSCs into the desired cell types in situ in the tissue, nanotechnology is introduced and used to facilitate or promote cell growth and differentiation. These nano-materials can provide a fine structure and tuneable surface in nanoscales to help the cell adhesion and promote the cell growth and differentiation of MSCs. This could be a dominant direction in future for stem cells based therapy or tissue engineering for various diseases. Therefore, the isolation, manipulation, and differentiation of MSCs are very important steps to make meaningful use of MSCs for disease treatments. In this chapter, we have described a method of isolating MSC from human bone marrow, and how to culture and differentiate them in vitro. We have also provided research methods on how to use MSCs in an in vitro model and how to observe MSC biological response on the surface of nano-scaled materials.

Immunosuppressive properties of mesenchymal stromal cell cultures derived from the limbus of human and rabbit corneas

Background aims Mesenchymal stromal cells (MSCs) cultivated from the corneal limbus (L-MSCs) provide a potential source of cells for corneal repair. In the present study, we investigated the immunosuppressive properties of human L-MSCs and putative rabbit L-MSCs to develop an allogeneic therapy and animal model of L-MSC transplantation. Methods MSC-like cultures were established from the limbal stroma of human and rabbit (New Zealand white) corneas using either serum-supplemented medium or a commercial serum-free MSC medium (MesenCult-XF Culture Kit; Stem Cell Technologies, Melbourne, Australia). L-MSC phenotype was examined by flow cytometry. The immunosuppressive properties of L-MSC cultures were assessed using mixed leukocyte reactions. L-MSC cultures were also tested for their ability to support colony formation by primary limbal epithelial (LE) cells. Results Human L-MSC cultures were typically CD34−, CD45− and HLA-DR− and CD73+, CD90+, CD105+ and HLA-ABC+. High levels (>80%) of CD146 expression were observed for L-MSC cultures grown in serum-supplemented medium but not cultures grown in MesenCult-XF (approximately 1%). Rabbit L-MSCs were approximately 95% positive for major histocompatibility complex class I and expressed lower levels of major histocompatibility complex class II (approximately 10%), CD45 (approximately 20%), CD105 (approximately 60%) and CD90 (<10%). Human L-MSCs and rabbit L-MSCs suppressed human T-cell proliferation by up to 75%. Conversely, L-MSCs from either species stimulated a 2-fold to 3-fold increase in LE cell colony formation. Conclusions L-MSCs display immunosuppressive qualities in addition to their established non-immunogenic profile and stimulate LE cell growth in vitro across species boundaries. These results support the potential use of allogeneic L-MSCs in the treatment of corneal disorders and suggest that the rabbit would provide a useful pre-clinical model.

Evaluation of canine bone marrow-derived mesenchymal stem cells after long-term cryopreservation

Autologous bone marrow-derived mesenchymal stem cell (BMSCs)-based therapies show great potential in regenerative medicine. However, long-term storage and preservation of BMSCs for clinical use is still a great clinical challenge. The present study aimed to analyze the effect of long-term cryopreservation on the regenerative ability of BMSCs. After cryopreservation of BMSCs from beagle dogs for three years, cell viability, and quantitative analysis of alkaline phosphatase (ALP) activity, surface adherence, and mineralized nodule formation were analyzed. BMSCs in cell-scaffold complex were then implanted into nude mice. There was no significant difference in cell viability and ALP activity between osteogenic differentiation and non-osteogenic differentiation of BMSCs, and BMSCs in cell-scaffold complex retained osteogenic differentiation ability in vivo. These results indicate that long-term cryopreserved BMSCs maintain their have capacity to contribute to regeneration.