A chimeric vitronectin : IGF-I protein supports feeder-cell-free and serum-free culture of human embryonic stem cells

This paper was retracted by the Journal of Stem Cells and Development on February 15, 2013.

Differences between in vitro viability and differentiation and in vivo bone-forming efficacy of human mesenchymal stem cells cultured on PCL-TCP scaffolds

Human mesenchymal stem cells (hMSCs) possess great therapeutic potential for the treatment of bone disease and fracture non-union. Too often however, in vitro evidence alone of the interaction between hMSCs and the biomaterial of choice is used as justification for continued development of the material into the clinic. Clearly for hMSC-based regenerative medicine to be successful for the treatment of orthopaedic trauma, it is crucial to transplant hMSCs with a suitable carrier that facilitates their survival, optimal proliferation and osteogenic differentiation in vitro and in vivo. This motivated us to evaluate the use of polycaprolactone-20% tricalcium phosphate (PCL-TCP) scaffolds produced by fused deposition modeling for the delivery of hMSCs. When hMSCs were cultured on the PCL-TCP scaffolds and imaged by a combination of phase contrast, scanning electron and confocal laser microscopy, we observed five distinct stages of colonization over a 21-day period that were characterized by cell attachment, spreading, cellular bridging, the formation of a dense cellular mass and the accumulation of a mineralized extracellular matrix when induced with osteogenic stimulants. Having established that PCL-TCP scaffolds are able to support hMSC proliferation and osteogenic differentiation, we next tested the in vivo efficacy of hMSC-loaded PCL-TCP scaffolds in nude rat critical-sized femoral defects. We found that fluorescently labeled hMSCs survived in the defect site for up to 3 weeks post-transplantation. However, only 50% of the femoral defects treated with hMSCs responded favorably as determined by new bone volume. As such, we show that verification of hMSC viability and differentiation in vitro is not sufficient to predict the efficacy of transplanted stem cells to consistently promote bone formation in orthotopic defects in vivo.

Cell sourcing for bone tissue engineering : amniotic fluid stem cells have a delayed, robust differentiation compared to mesenchymal stem cells

Cell based therapies for bone regeneration are an exciting emerging technology, but the availability of osteogenic cells is limited and an ideal cell source has not been identified. Amniotic fluid-derived stem (AFS) cells and bone-marrow derived mesenchymal stem cells (MSCs) were compared to determine their osteogenic differentiation capacity in both 2D and 3D environments. In 2D culture, the AFS cells produced more mineralized matrix but delayed peaks in osteogenic markers. Cells were also cultured on 3D scaffolds constructed of poly-e-caprolactone for 15 weeks. MSCs differentiated more quickly than AFS cells on 3D scaffolds, but mineralized matrix production slowed considerably after 5 weeks. In contrast, the rate of AFS cell mineralization continued to increase out to 15 weeks, at which time AFS constructs contained 5-fold more mineralized matrix than MSC constructs. Therefore, cell source should be taken into consideration when used for cell therapy, as the MSCs would be a good choice for immediate matrix production, but the AFS cells would continue robust mineralization for an extended period of time. This study demonstrates that stem cell source can dramatically influence the magnitude and rate of osteogenic differentiation in vitro.

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.

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.

The molecular mechanisms utilised by mesenchymal stem cells in migration to acute myocardial infarction

Heart damage caused by acute myocardial infarction (AMI) is a leading cause of death and disability in Australia. Novel therapies are still required for the treatment of this condition due to the poor reparative ability of the heart. As such, cellular therapies that assist in the recovery of heart muscle are of great current interest. Culture expanded mesenchymal stem cells (MSC) represent a stem and progenitor cell population that has been shown to promote tissue recovery in pre-clinical studies of AMI. For MSC-based therapies in the clinic, an intravenous route of administration would ideally be used due to the low cost, ease of delivery and relative safety. The study of MSC migration is therefore clinically relevant for a minimally invasive cell therapy to promote regeneration of damaged tissue. C57BL/6, UBI-GFP-BL/6 and CD44-/-/GFP+/+ mice were utilised to investigate mMSC migration. To assist in murine models of MSC migration, a novel method was used for the isolation of murine MSC (mMSC). These mMSC were then expanded in culture and putative mMSC were positive for Sca-1, CD90.2, and CD44 and were negative for CD45 and CD11b. Furthermore, mMSC from C57BL/6 and UBI-GFP-BL/6 mice were shown to differentiate into cells of the mesodermal lineage. Cells from CD44-/-/GFP+/+ mice were positive for Sca-1 and CD90.2, and negative for CD44, CD45 and CD11b however, these cells were unable to differentiate into adipocytes and chondrocytes and express lineage specific genes, PLIN and ACAN. Analysis of mMSC chemokine receptor (CR) expression showed that although mMSC do express chemokine receptors, (including those specific for chemokines released after AMI), these were low or undetectable by mRNA. However, protein expression could be detected, which was predominantly cytoplasmic. It was further shown that in both healthy (unperturbed) and inflamed tissues, mMSC had very little specific migration and engraftment after intravenous injection. To determine if poor mMSC migration was due to the inability of mMSC to respond to chemotactic stimuli, chemokine expression in bone marrow, skin injury and hearts (healthy and after AMI) was analysed at various time points by quantitative real-time PCR (qRT PCR). Many chemokines were up-regulated after skin biopsy and AMI, but the highest acute levels were found for CXCL12 and CCL7. Due to their high expression in infarcted hearts, the chemokines CXCL12 and CCL7 were tested for their effect on mMSC migration. Despite CR expression at both protein and mRNA levels, migration in response to CXCL12 and CCL7 was low in mMSC cultured on Nunclon plastic. A novel tissue culture plastic technology (UpCellTM) was then used that allowed gentle non-enzymatic dissociation of mMSC, thus preserving surface expression of the CRs. Despite this the in vitro data indicated that CXCL12 fails to induce significant migration ability of mMSC, while CCL7 induces significant, but low-level migration. We speculated this may be because of low levels of surface expression of chemokine receptors. In a strategy to increase cell surface expression of mMSC chemokine receptors and enhance their in vitro and in vivo migration capacity, mMSC were pre-treated with pro-inflammatory cytokines. Increased levels of both mRNA and surface protein expression were found for CRs by pre-treating mMSC with pro-inflammatory cytokines including TNF-á, IFN-ã, IL-1á and IL-6. Furthermore, the chemotactic response of mMSC to CXCL12 and CCL7 was significantly higher with these pretreated cells. Finally, the effectiveness of this type of cell manipulation was demonstrated in vivo, where mMSC pre-treated with TNF-á and IFN-ã showed significantly increased migration in skin injury and AMI models. Therefore this thesis has demonstrated, using in vitro and in vivo models, the potential for prior manipulation of MSC as a possible means for increasing the utility of intravenously delivery for MSC-based cellular therapies.

Monitoring mesenchymal stem cell cultures using image processing and pattern recognition techniques

Stem cells have attracted tremendous interest in recent times due to their promise in providing innovative new treatments for a great range of currently debilitating diseases. This is due to their potential ability to regenerate and repair damaged tissue, and hence restore lost body function, in a manner beyond the body's usual healing process. Bone marrow-derived mesenchymal stem cells or bone marrow stromal cells are one type of adult stem cells that are of particular interest. Since they are derived from a living human adult donor, they do not have the ethical issues associated with the use of human embryonic stem cells. They are also able to be taken from a patient or other donors with relative ease and then grown readily in the laboratory for clinical application. Despite the attractive properties of bone marrow stromal cells, there is presently no quick and easy way to determine the quality of a sample of such cells. Presently, a sample must be grown for weeks and subject to various time-consuming assays, under the direction of an expert cell biologist, to determine whether it will be useful. Hence there is a great need for innovative new ways to assess the quality of cell cultures for research and potential clinical application. The research presented in this thesis investigates the use of computerised image processing and pattern recognition techniques to provide a quicker and simpler method for the quality assessment of bone marrow stromal cell cultures. In particular, aim of this work is to find out whether it is possible, through the use of image processing and pattern recognition techniques, to predict the growth potential of a culture of human bone marrow stromal cells at early stages, before it is readily apparent to a human observer. With the above aim in mind, a computerised system was developed to classify the quality of bone marrow stromal cell cultures based on phase contrast microscopy images. Our system was trained and tested on mixed images of both healthy and unhealthy bone marrow stromal cell samples taken from three different patients. This system, when presented with 44 previously unseen bone marrow stromal cell culture images, outperformed human experts in the ability to correctly classify healthy and unhealthy cultures. The system correctly classified the health status of an image 88% of the time compared to an average of 72% of the time for human experts. Extensive training and testing of the system on a set of 139 normal sized images and 567 smaller image tiles showed an average performance of 86% and 85% correct classifications, respectively. The contributions of this thesis include demonstrating the applicability and potential of computerised image processing and pattern recognition techniques to the task of quality assessment of bone marrow stromal cell cultures. As part of this system, an image normalisation method has been suggested and a new segmentation algorithm has been developed for locating cell regions of irregularly shaped cells in phase contrast images. Importantly, we have validated the efficacy of both the normalisation and segmentation method, by demonstrating that both methods quantitatively improve the classification performance of subsequent pattern recognition algorithms, in discriminating between cell cultures of differing health status. We have shown that the quality of a cell culture of bone marrow stromal cells may be assessed without the need to either segment individual cells or to use time-lapse imaging. Finally, we have proposed a set of features, that when extracted from the cell regions of segmented input images, can be used to train current state of the art pattern recognition systems to predict the quality of bone marrow stromal cell cultures earlier and more consistently than human experts.