Bioreactors to influence stem cell fate: Augmentation of mesenchymal stem cell signaling pathways via dynamic culture systems

Background

Mesenchymal stem cells (MSCs) are a promising cell source for bone and cartilage tissue engineering as they can be easily isolated from the body and differentiated into osteoblasts and chondrocytes. A cell based tissue engineering strategy using MSCs often involves the culture of these cells on three-dimensional scaffolds; however the size of these scaffolds and the cell population they can support can be restricted in traditional static culture. Thus dynamic culture in bioreactor systems provides a promising means to culture and differentiate MSCs in vitro.

Scope of review

This review seeks to characterize key MSC differentiation signaling pathways and provides evidence as to how dynamic culture is augmenting these pathways. Following an overview of dynamic culture systems, discussion will be provided on how these systems can effectively modify and maintain important culture parameters including oxygen content and shear stress. Literature is reviewed for both a highlight of key signaling pathways and evidence for regulation of these signaling pathways via dynamic culture systems.

Major conclusions

The ability to understand how these culture systems are affecting MSC signaling pathways could lead to a shear or oxygen regime to direct stem cell differentiation. In this way the efficacy of in vitro culture and differentiation of MSCs on three-dimensional scaffolds could be greatly increased.

General significance

Bioreactor systems have the ability to control many key differentiation stimuli including mechanical stress and oxygen content. The further integration of cell signaling investigations within dynamic culture systems will lead to a quicker realization of the promise of tissue engineering and regenerative medicine. This article is part of a Special Issue entitled Biochemistry of Stem Cells.     

Secretion profile of human bone marrow stromal cells: Donor variability and response to inflammatory stimuli

Mesenchymal stem cells (MSC) derived from bone marrow are ideal transplants for a variety of CNS disorders and appear to support recovery after injury by secreting therapeutic factors. There is considerable variability in the secretion profile of MSC derived from different donors and it is known that MSC secretion changes in response to inflammatory stimuli, but no comprehensive analysis has been performed to address these issues. Here we show that MSC from seven donors secrete chemokines and cytokines in variable ranges, with some factors showing high variability. Treatment of cultured MSC with pro-inflammatory cytokines or tissue extracts from injured spinal cord resulted in up-regulation of selected cytokines, whereas treatment with an anti-inflammatory cytokine had little effect, indicating that the secretion profile is tightly regulated by environmental challenges. Patterns of up-regulated cytokines were similar in MSC from different donors suggesting a comparable response to inflammatory stimuli.     

In vitro mesenchymal stem cell differentiation after mechanical stimulation

Objectives: Mesenchymal stem cells (MSC) are multipotent cells capable of differentiating into adipocytic, chondrocytic and osteocytic lineages on suitable stimulation. We have hypothesized that mechanical loading may influence MSC differentiation and alter their phenotype accordingly. Materials and methods: Mouse bone marrow‐derived MSC were established in vitro by differential adherence to plastic culture plates and grown in low glucose medium with 10% foetal calf serum and growth factors. Cells grew out and were subcultured up to 20 times. Differentiation protocols were followed for several cell lineages. Clones with trilineage potential were seeded in type I collagen gels and incubated in a tensioning force bioreactor and real‐time cell‐derived forces were recorded. Gels were fixed and sectioned for light and electron microscopy. Results: Cell monolayers of parent and cloned mouse bone marrow‐derived MSC differentiated into adipocytes, osteocytes and chondrocytes, but not into cardiomyocytes, myotubes or neuronal cells. When cast into type I collagen gels and placed in tensioning bioreactors, MSC differentiated into fibroblast‐like cells typical of tissue stroma, and upregulated α‐smooth muscle actin, but rarely upregulated desmin. Electron microscopy showed collagen and elastin fibre synthesis into the matrix. Conclusions: These experiments confirmed that MSC cell fate choice depends on minute, cell‐derived forces. Applied force could assist in commercial manufacture of cultured bio‐engineered prostheses for regenerative medicine as it mimics tissue stresses and constitutes a good model for development of tissue substitutes.     

Functional properties of bone marrow-derived MSC-based engineered cartilage are unstable with very long-term in vitro culture

The success of stem cell-based cartilage repair requires that the regenerate tissue reach a stable state. To investigate the long-term stability of tissue engineered cartilage constructs, we assessed the development of compressive mechanical properties of chondrocyte and mesenchymal stem cell (MSC)-laden three dimensional agarose constructs cultured in a well defined chondrogenic in vitro environment through 112 days. Consistent with previous reports, in the presence of TGF-β, chondrocytes outperformed MSCs through day 56, under both free swelling and dynamic culture conditions, with MSC-laden constructs reaching a plateau in mechanical properties between days 28 and 56. Extending cultures through day 112 revealed that MSCs did not simply experience a lag in chondrogenesis, but rather that construct mechanical properties never matched those of chondrocyte-laden constructs. After 56 days, MSC-laden constructs underwent a marked reversal in their growth trajectory, with significant declines in glycosaminoglycan content and mechanical properties. Quantification of viability showed marked differences in cell health between chondrocytes and MSCs throughout the culture period, with MSC-laden construct cell viability falling to very low levels at these extended time points. These results were not dependent on the material environment, as similar findings were observed in a photocrosslinkable hyaluronic acid (HA) hydrogel system that is highly supportive of MSC chondrogenesis. These data suggest that, even within a controlled in vitro environment that is conducive to chondrogenesis, there may be an innate instability in the MSC phenotype that is independent of scaffold composition, and may ultimately limit their application in functional cartilage repair.     

Mesenchymal stem cell secretome and regenerative therapy after cancer

Cancer treatment generally relies on tumor ablative techniques that can lead to major functional or disfiguring defects. These post-therapy impairments require the development of safe regenerative therapy strategies during cancer remission. Many current tissue repair approaches exploit paracrine (immunomodulatory, pro-angiogenic, anti-apoptotic and pro-survival effects) or restoring (functional or structural tissue repair) properties of mesenchymal stem/stromal cells (MSC). Yet, a major concern in the application of regenerative therapies during cancer remission remains the possible triggering of cancer recurrence. Tumor relapse implies the persistence of rare subsets of tumor-initiating cancer cells which can escape anti-cancer therapies and lie dormant in specific niches awaiting reactivation via unknown stimuli. Many of the components required for successful regenerative therapy (revascularization, immunosuppression, cellular homing, tissue growth promotion) are also critical for tumor progression and metastasis. While bi-directional crosstalk between tumorigenic cells (especially aggressive cancer cell lines) and MSC (including tumor stroma-resident populations) has been demonstrated in a variety of cancers, the effects of local or systemic MSC delivery for regenerative purposes on persisting cancer cells during remission remain controversial. Both pro- and anti-tumorigenic effects of MSC have been reported in the literature. Our own data using breast cancer clinical isolates have suggested that dormant-like tumor-initiating cells do not respond to MSC signals, unlike actively dividing cancer cells which benefited from the presence of supportive MSC. The secretome of MSC isolated from various tissues may partially diverge, but it includes a core of cytokines (i.e. CCL2, CCL5, IL-6, TGFβ, VEGF), which have been implicated in tumor growth and/or metastasis. This article reviews published models for studying interactions between MSC and cancer cells with a focus on the impact of MSC secretome on cancer cell activity, and discusses the implications for regenerative therapy after cancer.     

Culture media conditioned by heat-shocked osteoblasts enhances the osteogenesis of bone marrow-derived mesenchymal stromal cells

Osteogenic cells differentiated from bone marrow-derived mesenchymal stromal cells (MSC) hold much promise in bone tissue engineering and reconstructive surgery. There is a dire need for well-defined and efficient protocols to promote the osteogenesis of ex vivo cultured MSC. Hence, this study investigated whether a combination of chemical stimuli (ascorbic acid, beta-glycerophosphate and dexamethasone) and culture media conditioned by a human foetal osteoblast cell line (hFOB) had any synergistic effect on the osteogenesis of MSC. Conditioned media with or without prior heat shock treatment (42 degrees C for 1 h) of the hFOB cell line, were collected and tested on rabbit MSC cultures, in the presence and absence of chemical stimuli. Osteogenic differentiation of MSC was assessed on both day 14 and 21 of ex vivo culture. The results showed conclusively that conditioned media promoted osteogenesis of MSC, which was further enhanced by prior heat shock-treatment of the hFOB cells, as well as by the presence of chemical stimuli. Among all experimental groups, the combination of culture medium conditioned by heat shocked hFOB cells together with chemical stimuli, exhibited the highest level of calcium mineralization, as assessed by Von Kossa staining. This provides clear evidence of a synergistic effect of conditioned media, heat shock and chemical stimuli. It is hoped that the data may contribute to the development of a more well-defined and efficient in vitro culture protocol to promote the osteogenesis of MSC for both clinical and non-clinical applications.     

Regulation of differentiation of mesenchymal stem cells into musculoskeletal cells

Mesenchymal stem cells (MSCs) are multipotent cells that have the capability of differentiating into several different cells such as osteoblasts (bone), chondrocytes (cartilage), adipocytes (fat), myocytes (muscle) and tenocytes (tendon). In this review we highlight the different regulators which determine the lineage a particular MSC will differentiate into. Mesenchymal stem cells are increasingly being used in tissue regeneration and repair. Strict regulation of differentiation of MSCs is essential for a positive outcome of the particular tissue treated with MSCs, especially due to the fact that capacity to differentiate decreases with increasing age of the donor.     

Mesenchymal stem cells: biological characteristics and potential clinical applications

Mesenchymal stem cells (MSC) are clonogenic, non-hematpoietic stem cells present in the bone marrow and are able to differentiate into multiple mesoderm-type cell lineages, for example, osteoblasts, chondrocytes, endothelial-cells and also non-mesoderm-type lineages, for example, neuronal-like cells. Several methods are currently available for isolation of the MSC based on their physical and physico-chemical characteristics, for example, adherence to plastics or other extracellular matrix components. Because of the ease of their isolation and their extensive differentiation potential, MSC are among the first stem cell types to be introduced in the clinic. Several studies have demonstrated the possible use of MSC in systemic transplantation for systemic diseases, local implantation for local tissue defects, as a vehicle for genes in gene therapy protocols or to generate transplantable tissues and organs in tissue engineering protocols. Before their widespread use in therapy, methods allowing the generation of large number of cells without affecting their differentiation potential as well as technologies that overcome immunological rejection (in case allogenic transplantation) must be developed.     

Expression patterns and function of chromatin protein HMGB2 during mesenchymal stem cell differentiation

The superficial zone (SZ) of articular cartilage is critical in maintaining tissue function and homeostasis and represents the site of the earliest changes in osteoarthritis (OA). The expression of chromatin protein HMGB2 is restricted to the SZ, which contains cells expressing mesenchymal stem cell (MSC) markers. Age-related loss of HMGB2 and gene deletion are associated with reduced SZ cellularity and early onset OA. This study addressed HMGB2 expression patterns in MSC and its role during differentiation. HMGB2 was detected at higher levels in human MSC as compared with human articular chondrocytes, and its expression declined during chondrogenic differentiation of MSC. Lentiviral HMGB2 transduction of MSC suppressed chondrogenesis as reflected by an inhibition of Col2a1 and Col10a1 expression. Conversely, in bone marrow MSC from Hmgb2(-/-) mice, Col10a1 was more strongly expressed than in wild-type MSC. This is consistent with in vivo results from mouse growth plates showing that Hmgb2 is expressed in proliferating and prehypertrophic zones but not in hypertrophic cartilage where Col10a1 is strongly expressed. Osteogenesis was also accelerated in Hmgb2(-/-) MSC. The expression of Runx2, which plays a major role in late stage chondrocyte differentiation, was enhanced in Hmgb2(-/-) MSC, and HMGB2 negatively regulated the stimulatory effect of Wnt/β-catenin signaling on the Runx2 proximal promoter. These results demonstrate that HMGB2 expression is inversely correlated with the differentiation status of MSC and that HMGB2 suppresses chondrogenic differentiation. The age-related loss of HMGB2 in articular cartilage may represent a mechanism responsible for the decline in adult cartilage stem cell populations.     

Mechanical responses and integrin associated protein expression by human ankle chondrocytes

Metabolic, biochemical and biomechanical differences between ankle and knee joint cartilage and chondrocytes including resistance to the effects of catabolic cytokines and fibronectin fragments may be relevant to differences in prevalence of OA in these joints. Although there is increasing information available on how chondrocytes from knee and hip joint cartilage recognise and respond to mechanical stimuli, knowledge of mechanotransduction in ankle joint chondrocytes is limited. This study was undertaken to (i) establish whether the response of normal ankle joint derived chondrocytes to mechanical stimulation in vitro was similar to that of normal and osteoarthritic knee joint derived chondrocytes and (ii) to investigate whether these chondrocytes showed differences in expression of integrin associated regulatory and signalling molecules. Unlike normal knee joint chondrocytes, ankle joint chondrocytes did not show an increase in relative levels of aggrecan mRNA when mechanically stimulated. No obvious change in protein tyrosine phosphorylation was seen in ankle chondrocytes subsequent to mechanical stimulation but these cells expressed elevated levels of tyrosine phosphorylated proteins at rest when compared to normal knee joint chondrocytes. Ankle joint chondrocytes showed an increase in protein kinase B phosphorylation following 1 min 0.33 Hz stimulation which was inhibited by the presence of antibodies to alpha5beta1 integrin. Ankle joint chondrocytes appeared to show significant differences in levels of the integrin-associated proteins CD98, CD147 and galectin 3, PKCgamma and differences in responses to glutamate were seen. Chondrocytes from ankle and knee joint cartilage respond differently to 0.33 Hz mechanical stimulation. This may be related to modified integrin-dependent mechanotransduction as a result of changes in expression of integrin regulatory molecules such as CD98 or differential expression and function of downstream components of the mechanotransduction pathway such as PKC or NMDA receptors.     

Comparative analysis of cell populations with a phenotype similar to that of mesenchymal stem cells derived from subcutaneous fat

Two cell populations with a phenotype similar to that of mesenchymal stem cells (MSC) with different characteristics for expression of surface antigene CD34 were derived from subcutaneous fat. CD34-positive cells were derived from subcutaneous fat of the inferior eyelid obtained during transconjuctival blepharoplasty. CD34-negative cells were derived from adipose tissue obtained during lipoaspiration from the same patients. These cells displayed common characteristics for morphology and expression of basic markers characterizing them as mesenchymal stem cells. On being induced for differentiation, these two cell populations were able to differentiate to cells of adipose (adipocytes), bone (osteoblastes, osteocytes), cartilage (chondroblasts, chondrocytes), and nervous (neurons, astrocytes and oligodendrocytes) tissues.     

Mesenchymal stem cells: From bench to bedside

Human mesenchymal stem cells (hMSCs) have tremendous promise for use in a variety of clinical applications. The ability of these cells to self-renew and differentiate into multiple tissues makes them an attractive cell source for a new generation of cell-based regenerative therapies. Encouraging results from clinical trials have also generated growing enthusiasm regarding MSC therapy and related treatment, but gaps remain in understanding MSC tissue repair mechanisms and in clinical strategies for efficient cell delivery and consistent therapeutic outcomes. For these reasons, discoveries from basic research and their implementation in clinical trials are essential to advance MSC therapy from the laboratory bench to the patient's bedside.     

Mesenchymal stem cells as a potent cell source for articular cartilage regeneration

Since articular cartilage possesses only a weak capac-ity for repair, its regeneration potential is considered one of the most important challenges for orthopedic surgeons. The treatment options, such as marrow stimulation techniques, fail to induce a repair tissue with the same functional and mechanical properties of native hyaline cartilage. Osteochondral transplantation is considered an effective treatment option but is as-sociated with some disadvantages, including donor-site morbidity, tissue supply limitation, unsuitable mechani-cal properties and thickness of the obtained tissue. Although autologous chondrocyte implantation results in reasonable repair, it requires a two-step surgical pro-cedure. Moreover, chondrocytes expanded in culture gradually undergo dedifferentiation, so lose morpho-logical features and specialized functions. In the search for alternative cells, scientists have found mesenchymal stem cells(MSCs) to be an appropriate cellular mate-rial for articular cartilage repair. These cells were origi-nally isolated from bone marrow samples and further investigations have revealed the presence of the cells in many other tissues. Furthermore, chondrogenic dif-ferentiation is an inherent property of MSCs noticedat the time of the cell discovery. MSCs are known to exhibit homing potential to the damaged site at which they differentiate into the tissue cells or secrete a wide spectrum of bioactive factors with regenerative proper-ties. Moreover, these cells possess a considerable im-munomodulatory potential that make them the general donor for therapeutic applications. All of these topics will be discussed in this review.     

Allogeneic mesenchymal stem cells: agents of immune modulation

Adult mesenchymal stem cells possess a remarkably diverse array of immunosuppressive characteristics. The capacity to suppress the regular processes of allogeneic rejection, have allowed the use of tissue mismatched cells as therapeutic approaches in regenerative medicine and as agents of immune deviation. This review describes recent advances in understanding the mechanistic basis of mesenchymal stromal or stem cells (MSC) interaction with innate immunity. Particular emphasis is placed on the effect of Toll-like receptor signalling on MSC and a hypothesis that innate immune signals induce a 'licensing switch' in MSC is put forward. The mechanisms underlying MSC suppression of T cell responses and induction of regulatory populations are surveyed. Conflicting data regarding the influence of MSC on B cell function are outlined and discussed. Finally the limits to MSC mediated immune modulation are discussed with reference to the future clinical application of novel cell therapies.     

Morphogenetic signals from chondrocytes promote chondrogenic and osteogenic differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are potentially useful cells for musculoskeletal tissue engineering. However, controlling MSC differentiation and tissue formation in vivo remains a challenge. There is a significant need for well-defined and efficient protocols for directing MSC behaviors in vivo. We hypothesize that morphogenetic signals from chondrocytes may regulate MSC differentiation. In micromass culture of MSCs, incubation with chondrocyte-conditioned medium (CCM) significantly enhanced the production of cartilage specific matrix including type II collagen. In addition, incubation of MSCs with conditioned medium supplemented with osteogenic factors induced more osteogenesis and accumulation of calcium and increased ALP activity. These findings reveal that chondrocyte-secreted factors promote chondrogenesis as well as osteogenesis of MSCs during in vitro micromass culture. Moreover, when MSCs expanded with chondrocyte-conditioned medium were encapsulated in hydrogels and subsequently implanted into athymic mice, basophilic extracellular matrix deposition characteristic of neocartilage was evident. These results indicate that articular chondrocytes produce suitable morphogenetic factors that induce the differentiation program of MSCs in vitro and in vivo.     

Engineered Co-culture Strategies Using Stem Cells for Facilitated Chondrogenic Differentiation and Cartilage Repair

Osteoarthritis (OA) is a chronic disease in elders and athletes due to limited regenerative capacities of cartilage tissues and subsequently insufficient recovery of damaged sites. Recent clinical treatments for OA have utilized progenitor cell-based therapies for cartilage tissue regeneration. Administration of a single type of cell population such as stem cells or chondrocytes does not guarantee a full recovery of cartilage defects. Therefore, current tissue engineering approaches using co-culture techniques have been developed to mimic complex and dynamic cellular interactions in native cartilage tissues and facilitate changes in cellular phenotypes into chondrogenesis. Therefore, this paper introduces recently developed co-culture systems using two major cell populations, mesenchymal stem cells (MSCs) and chondrocytes. Specifically, a series of examples to describe (1) synergistic in vitro activations of MSCs by paracrine signaling molecules from adult chondrocytes in co-culture systems and (2) functional in vivo tissue regeneration via co-administration of both cell types were reviewed. Based on these findings, it could be speculated that engineered co-culture systems using MSC/ chondrocyte is a promising and feasible cell-based OA therapy in clinical aspects.     

Mesenchymal stem cell durotaxis depends on substrate stiffness gradient strength

Mesenchymal stem cells (MSCs) respond to the elasticity of their environment, which varies between and within tissues. Stiffness gradients within tissues can result from pathological conditions, but also occur through normal variation, such as in muscle. MSC migration can be directed by shallow stiffness gradients before differentiating. Gradients with fine control over substrate compliance – both in range and rate of change (strength) – are needed to better understand mechanical regulation of MSC migration in normal and diseased states. We describe polyacrylamide stiffness gradient fabrication using three distinct systems, generating stiffness gradients of physiological (1 Pa/μm), pathological (10 Pa/μm), and step change (≥ 100Pa/μm) strength. All gradients spanned a range of physiologically relevant elastic moduli for soft tissues (1–12 kPa). MSCs migrated to the stiffest region on each gradient. Time-lapse microscopy revealed that migration velocity correlated directly with gradient strength. Directed migration was reduced in the presence of the contractile agonist lysophosphatidic acid (LPA) and cytoskeleton-perturbing drugs nocodazole and cytochalasin. LPA- and nocodazole-treated cells remained spread and protrusive on the substrate, while cytochalasin-treated cells did not. Nocodazole-treated cells spread in a similar manner to untreated cells, but exhibited greatly diminished traction forces. These data suggest that a functional actin cytoskeleton is required for migration whereas microtubules are required for directed migration. The data also imply that, in vivo, MSCs may preferentially accumulate in regions of high elastic modulus and make a greater contribution to tissue repairs in these locations.     

Repair of injured articular and growth plate cartilage using mesenchymal stem cells and chondrogenic gene therapy

Injuries to the articular cartilage and growth plate are significant clinical problems due to their limited ability to regenerate themselves. Despite progress in orthopedic surgery and some success in development of chondrocyte transplantation treatment and in early tissue-engineering work, cartilage regeneration using a biological approach still remains a great challenge. In the last 15 years, researchers have made significant advances and tremendous progress in exploring the potentials of mesenchymal stem cells (MSCs) in cartilage repair. These include (a) identifying readily available sources of and devising appropriate techniques for isolation and culture expansion of MSCs that have good chondrogenic differentiation capability, (b) discovering appropriate growth factors (such as TGF-beta, IGF-I, BMPs, and FGF-2) that promote MSC chondrogenic differentiation, (c) identifying or engineering biological or artificial matrix scaffolds as carriers for MSCs and growth factors for their transplantation and defect filling. In addition, representing another new perspective for cartilage repair is the successful demonstration of gene therapy with chondrogenic growth factors or inflammatory inhibitors (either individually or in combination), either directly to the cartilage tissue or mediated through transducing and transplanting cultured chondrocytes, MSCs or other mesenchymal cells. However, despite these rapid pre-clinical advances and some success in engineering cartilage-like tissue and in repairing articular and growth plate cartilage, challenges of their clinical translation remain. To achieve clinical effectiveness, safety, and practicality of using MSCs for cartilage repair, one critical investigation will be to examine the optimal combination of MSC sources, growth factor cocktails, and supporting carrier matrixes. As more insights are acquired into the critical factors regulating MSC migration, proliferation and chondrogenic differentiation both ex vivo and in vivo, it will be possible clinically to orchestrate desirable repair of injured articular and growth plate cartilage, either by transplanting ex vivo expanded MSCs or MSCs with genetic modifications, or by mobilising endogenous MSCs from adjacent source tissues such as synovium, bone marrow, or trabecular bone.