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The development of the musculoskeletal system is a complex process that involves very precise control of bone formation and growth as well as remodeling during postnatal life. Although the understanding of the transcriptional mechanisms of osteogenesis has increased considerably, the molecular regulatory basis, especially the gene regulatory network of osteogenic differentiation, is still poorly understood. This review provides the reader with an overview of the key transcription factors that govern bone formation, highlighting their function and regulation linked to Runt-related transcription factor 2 (Runx2). Runx2 as the master transcription factor of osteoblast differentiation, Twist, Msh homeobox 2 (Msx2), and promyelocytic leukemia zinc-finger protein (PLZF) acting upstream of Runx2, Osterix (Osx) acting downstream of Runx2, and activating transcription factor 4 (ATF4) and zinc-finger protein 521 (ZFP521) acting as cofactors of Runx2 are discussed, and their relevance for tissue engineering is presented. References are provided for more in-depth personal study.

Mesenchymal stromal cells (MSCs) are promising therapeutic agents for cartilage regeneration, including the potential of cells to promote chondrogenesis in vivo. However, process development and regulatory approval of MSCs as cell therapy products benefit from facile in vitro approaches that can predict potency for a given production run. Current standard in vitro approaches include a 21 day 3D differentiation assay followed by quantification of cartilage matrix proteins. We propose a novel biophysical marker that is cell population-based and can be measured from in vitro monolayer culture of MSCs. We hypothesized that the self-assembly pattern that emerges from collective-cell behavior would predict chondrogenesis motivated by our observation that certain features in this pattern, namely, topological defects, corresponded to mesenchymal condensations. Indeed, we observed a strong predictive correlation between the degree-of-order of the pattern at day 9 of the monolayer culture and chondrogenic potential later estimated from in vitro 3D chondrogenic differentiation at day 21. These findings provide the rationale and the proof-of-concept for using self-assembly patterns to monitor chondrogenic commitment of cell populations. Such correlations across multiple MSC donors and production batches suggest that self-assembly patterns can be used as a candidate biophysical attribute to predict quality and efficacy for MSCs employed therapeutically for cartilage regeneration.

Background Secretome profiles of mesenchymal stem cells (MSCs) are reflective of their local microenvironments. These biologically active factors exert an impact on the surrounding cells, eliciting regenerative responses that create an opportunity for exploiting MSCs towards a cell-free therapy for cartilage regeneration. The conventional method of culturing MSCs on a tissue culture plate (TCP) does not provide the physiological microenvironment for optimum secretome production. In this study, we explored the potential of electrospun fiber sheets with specific orientation in influencing the MSC secretome production and its therapeutic value in repairing cartilage. Methods Conditioned media (CM) were generated from MSCs cultured either on TCP or electrospun fiber sheets of distinct aligned or random fiber orientation. The paracrine potential of CM in affecting chondrogenic differentiation, migration, proliferation, inflammatory modulation, and survival of MSCs and chondrocytes was assessed. The involvement of FAK and ERK mechanotransduction pathways in modulating MSC secretome were also investigated. Results We showed that conditioned media of MSCs cultured on electrospun fiber sheets compared to that generated from TCP have improved secretome yield and profile, which enhanced the migration and proliferation of MSCs and chondrocytes, promoted MSC chondrogenesis, mitigated inflammation in both MSCs and chondrocytes, as well as protected chondrocytes from apoptosis. Amongst the fiber sheet-generated CM, aligned fiber-generated CM (ACM) was better at promoting cell proliferation and augmenting MSC chondrogenesis, while randomly oriented fiber-generated CM (RCM) was more efficient in mitigating the inflammation assault. FAK and ERK signalings were shown to participate in the modulation of MSC morphology and its secretome production. Conclusions This study demonstrates topographical-dependent MSC paracrine activities and the potential of employing electrospun fiber sheets to improve the MSC secretome for cartilage regeneration.

Background The mesenchymal stem cell (MSC) secretome, via the combined actions of its plethora of biologically active factors, is capable of orchestrating the regenerative responses of numerous tissues by both eliciting and amplifying biological responses within recipient cells. MSCs are “environmentally responsive” to local micro-environmental cues and biophysical perturbations, influencing their differentiation as well as secretion of bioactive factors. We have previously shown that exposures of MSCs to pulsed electromagnetic fields (PEMFs) enhanced MSC chondrogenesis. Here, we investigate the influence of PEMF exposure over the paracrine activity of MSCs and its significance to cartilage regeneration. Methods Conditioned medium (CM) was generated from MSCs subjected to either 3D or 2D culturing platforms, with or without PEMF exposure. The paracrine effects of CM over chondrocytes and MSC chondrogenesis, migration and proliferation, as well as the inflammatory status and induced apoptosis in chondrocytes and MSCs was assessed. Results We show that benefits of magnetic field stimulation over MSC-derived chondrogenesis can be partly ascribed to its ability to modulate the MSC secretome. MSCs cultured on either 2D or 3D platforms displayed distinct magnetic sensitivities, whereby MSCs grown in 2D or 3D platforms responded most favorably to PEMF exposure at 2 mT and 3 mT amplitudes, respectively. Ten minutes of PEMF exposure was sufficient to substantially augment the chondrogenic potential of MSC-derived CM generated from either platform. Furthermore, PEMF-induced CM was capable of enhancing the migration of chondrocytes and MSCs as well as mitigating cellular inflammation and apoptosis. Conclusions The findings reported here demonstrate that PEMF stimulation is capable of modulating the paracrine function of MSCs for the enhancement and re-establishment of cartilage regeneration in states of cellular stress. The PEMF-induced modulation of the MSC-derived paracrine function for directed biological responses in recipient cells or tissues has broad clinical and practical ramifications with high translational value across numerous clinical applications.

Stem cell differentiation is guided by contact with the physical microenvironment, influence by both topography and mechanical properties of the matrix. In this study, the combined effect of substratum nano-topography and mechanical stiffness in directing mesenchymal stem cell (MSC) chondrogenesis was investigated. Three polyesters of varying stiffness were thermally imprinted to create nano-grating or pillar patterns of the same dimension. The surface of the nano-patterned substrate was coated with chondroitin sulfate (CS) to provide an even surface chemistry, with cell-adhesive and chondro-inductive properties, across all polymeric substrates. The surface characteristic, mechanical modulus, and degradation of the CS-coated patterned polymeric substrates were analyzed. The cell morphology adopted on the nano-topographic surfaces were accounted by F-actin distribution, and correlated to the cell proliferation and chondrogenic differentiation outcomes. Results show that substratum stiffness and topographical cues affected MSC morphology and aggregation, and influenced the phenotypic development at the earlier stage of chondrogenic differentiation. Hyaline-like cartilage with middle/deep zone cartilage characteristics was generated on softer pillar surface, while on stiffer nano-pillar material MSCs showed potential to generate constituents of hyaline/fibro/hypertrophic cartilage. Fibro/superficial zone-like cartilage could be derived from nano-grating of softer stiffness, while stiffer nano-grating resulted in insignificant chondrogenesis. This study demonstrates the possibility of refining the phenotype of cartilage generated from MSCs by manipulating surface topography and material stiffness.

Human mesenchymal stem cells (MSCs) have been studied in over 1500 clinical trials to treat over 30 diseases. However, the understanding towards MSC stemness remains under studied. So far, little is known about how MSCs maintain undifferentiated state or commit to specific lineages under different microenvironmental cues. The lack of comprehensive understanding regarding MSC stemness greatly hampers the translation of research findings into successful clinical application due to unclear mechanism of action. Emerging evidence shows that a variety of genetic factors delicately regulate MSC self-renewal and differentiation. In this review, we summarize the role of transcriptional factors, cell cycle regulators, genomic stability genes, cellular quality control, epigenetic regulators, non-coding RNAs, mitochondrial function, growth factors and m6A modification in regulating the stemness of MSCs. Strategies to maintain MSC stemness during ex-vivo expansion are also discussed. This review will deepen understanding of MSC stemness for advancing clinical applications and provide insights into future directions for research aimed at improving MSC-based therapies.

Articular cartilage defects commonly result from trauma and are associated with significant morbidity. Since cartilage is an avascular, aneural, and alymphatic tissue with a poor intrinsic healing ability, the regeneration of functional hyaline cartilage remains a difficult clinical problem. Mesenchymal stem cells (MSCs) are multipotent cells with multilineage differentiation potential, including the ability to differentiate into chondrocytes. Due to their availability and ease of ex vivo expansion, clinicians are increasingly applying MSCs in the treatment of cartilage lesions. However, despite encouraging pre-clinical and clinical data, inconsistencies in MSC proliferative and chondrogenic potential depending on donor, tissue source, cell subset, culture conditions, and handling techniques remain a key barrier to widespread clinical application of MSC therapy in cartilage regeneration. In this review, we highlight the strategies to manage the heterogeneity of MSCs ex vivo for more effective cartilage repair, including reducing the MSC culture expansion period, and selecting MSCs with higher chondrogenic potential through specific genetic markers, surface markers, and biophysical attributes. The accomplishment of a less heterogeneous population of culture-expanded MSCs may improve the scalability, reproducibility, and standardisation of MSC therapy for clinical application in cartilage regeneration.

Stem cell biology has gained tremendous interest in the recent years, driven by the hope of finding cures for several diseases through transplantation medicine. Various types of stem cells have been identified from preimplantation embryos, the fetus, placenta and adult. The isolation of human embryonic stem cells has been considered the biggest breakthrough of the 21st century. Some of these stem cell types are charged with ethical controversies although they are versatile and offer tremendous potential for finding cures for incurable diseases. In fact, cures for diabetes, heart diseases, Parkinson's, Alzheimer's, and many other diseases via stem cell research is expected within the next 10 years. The documentation of the latest information on these various stem cell types is compartmentalized in different journals and texts. This book brings together the state-of-the-art on all the types of stem cells, written by giants in the respective fields. It is hoped that knowledge of one stem cell type will complement the other and much can be learned by interaction. This book is aimed at undergraduates, postgraduates, scientists, embryologists, tissue engineers, doctors and biomedical scientists with an interest in stem cell research. It is different from other texts in that it is very comprehensive, covering all aspects of stem cells. Additionally, all the various chapters are written by renowned scientists in the specific fields. The topics are uniquely treated in that they cover research at the laboratory bench as well as the clinical applications. Many of the chapters carry extensive self-explanatory figures, colored photographs, graphics and tables. Both the editors are also renowned in the stem cell field, and both come from medical disciplines and are senior university academics with excellent track records.

Human embryonic stem cells (hESCs) are considered as useful tools for pre‐clinical studies in regenerative medicine. Although previous reports have shown direct chondrogenic differentiation of mouse and hESCs, low yield and cellular heterogenicity of the resulting cell population impairs the generation of sufficient numbers of differentiated cells for further testing and applications. Based on our previously established high‐density micromass model system to study hESC chondrogenesis, we evaluated the effects of transforming growth factor (TGF)‐β1 and bone morphogenetic protein‐2 on early stages of chondrogenic differentiation and commitment by hESCs. Significant chondrogenic induction of hESCs, as determined by quantitative measurements of cartilage‐related gene expression and matrix protein synthesis, was achieved in the presence of TGF‐β1. By means of selective growth factor combination (TGF‐β1, FGF‐2 and platelet‐derived growth factor‐bb) and plating on extracellular matrix substratum, we report here the reproducible isolation of a highly expandable, homogenous and unipotent chondrogenic cell population, TC1, from chondrogenically committed hESCs. Like primary chondrocytes, TC1 rapidly dedifferentiates upon isolation and monolayer expansion but retains the chondrogenic differentiation potential and responds to TGF‐β1 for cartilaginous tissue formation both in vitro and in vivo. In addition, TC1 displays a somatic cell cycle kinetics, a normal karyotype and does not produce teratoma in vivo. Thus, TC1 may provide a potential source of chondrogenic cells for drug testing, gene therapy and cell‐based therapy.