Induction of chondro-, osteo- and adipogenesis in embryonic stem cells by bone morphogenetic protein-2: Effect of cofactors on differentiating lineages

BACKGROUND: Recently, tissue engineering has merged with stem cell technology with interest to develop new sources of transplantable material for injury or disease treatment. Eminently interesting, are bone and joint injuries/disorders because of the low self-regenerating capacity of the matrix secreting cells, particularly chondrocytes. ES cells have the unlimited capacity to self-renew and maintain their pluripotency in culture. Upon induction of various signals they will then differentiate into distinctive cell types such as neurons, cardiomyocytes and osteoblasts. RESULTS: We present here that BMP-2 can drive ES cells to the cartilage, osteoblast or adipogenic fate depending on supplementary co-factors. TGFβ(1), insulin and ascorbic acid were identified as signals that together with BMP-2 induce a chondrocytic phenotype that is characterized by increased expression of cartilage marker genes in a timely co-ordinated fashion. Expression of collagen type IIB and aggrecan, indicative of a fully mature state, continuously ascend until reaching a peak at day 32 of culture to approximately 80-fold over control values. Sox9 and scleraxis, cartilage specific transcription factors, are highly expressed at very early stages and show decreased expression over the time course of EB differentiation. Some smaller proteoglycans, such as decorin and biglycan, are expressed at earlier stages. Overall, proteoglycan biosynthesis is up-regulated 7-fold in response to the supplements added. BMP-2 induced chondrocytes undergo hypertrophy and begin to alter their expression profile towards osteoblasts. Supplying mineralization factors such as β-glycerophosphate and vitamin D(3 )with the culture medium can facilitate this process. Moreover, gene expression studies show that adipocytes can also differentiate from BMP-2 treated ES cells. CONCLUSIONS: Ultimately, we have found that ES cells can be successfully triggered to differentiate into chondrocyte-like cells, which can further alter their fate to become hypertrophic, and adipocytes. Compared with previous reports using a brief BMP-2 supplementation early in differentiation, prolonged exposure increased chondrogenic output, while supplementation with insulin and ascorbic acid prevented dedifferentiation. These results provide a foundation for the use of ES cells as a potential therapy in joint injury and disease

Carboxyl-modified single-wall carbon nanotubes improve bone tissue formation in vitro and repair in an in vivo rat model.

The clinical management of bone defects caused by trauma or nonunion fractures remains a challenge in orthopedic practice due to the poor integration and biocompatibility properties of the scaffold or implant material. In the current work, the osteogenic properties of carboxyl-modified single-walled carbon nanotubes (COOH-SWCNTs) were investigated in vivo and in vitro. When human preosteoblasts and murine embryonic stem cells were cultured on coverslips sprayed with COOH-SWCNTs, accelerated osteogenic differentiation was manifested by increased expression of classical bone marker genes and an increase in the secretion of osteocalcin, in addition to prior mineralization of the extracellular matrix. These results predicated COOH-SWCNTs' use to further promote osteogenic differentiation in vivo. In contrast, both cell lines had difficulties adhering to multi-walled carbon nanotube-based scaffolds, as shown by scanning electron microscopy. While a suspension of SWCNTs caused cytotoxicity in both cell lines at levels >20 μg/mL, these levels were never achieved by release from sprayed SWCNTs, warranting the approach taken. In vivo, human allografts formed by the combination of demineralized bone matrix or cartilage particles with SWCNTs were implanted into nude rats, and ectopic bone formation was analyzed. Histological analysis of both types of implants showed high permeability and pore connectivity of the carbon nanotube-soaked implants. Numerous vascularization channels appeared in the formed tissue, additional progenitor cells were recruited, and areas of de novo ossification were found 4 weeks post-implantation. Induction of the expression of bone-related genes and the presence of secreted osteopontin protein were also confirmed by quantitative polymerase chain reaction analysis and immunofluorescence, respectively. In summary, these results are in line with prior contributions that highlight the suitability of SWCNTs as scaffolds with high bone-inducing capabilities both in vitro and in vivo, confirming them as alternatives to current bone-repair therapies

Embryonic stem cell therapy for osteo-degenerative diseases: Methods and protocols

Embryonic stem cells (ESCs) offer an unlimited self-renewing capacity, as opposed to the limits of adult stem cells; therefore, ESCs represent an almost bottomless resource for regenerative medicine and tissue engineering approaches. In Embryonic Stem Cell Therapy for Osteo-Degenerative Diseases: Methods and Protocols, accomplished investigators provide detailed descriptions on how to expand ESCs from the most commonly used species ex vivo, i.e. mouse and human, in static culture as well as in controllable bioreactor processes. The thorough and timely volume summarizes the methods that may be used to differentiate these cells along the desired lineage of choice, be it osteoblasts, osteoclasts, or chondrocytes, and consequentially also offers analysis tools for the characterization of resulting cells and evaluation of differentiation effectiveness. Written in the highly successful Methods in Molecular BiologyTM series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and expert tips on troubleshooting and avoiding known pitfalls. Dependable and cutting-edge, Embryonic Stem Cell Therapy for Osteo-Degenerative Diseases: Methods and Protocols supplies the tools necessary to allow researchers to carry out critical research needed in order to bring this burgeoning and vitally important field closer to the clinic and to ensure the widespread application of a successful strategy

microRNA Regulation of Skeletal Development

Purpose of reviewOsteogenesis is a complex process involving the specification of multiple progenitor cells and their maturation and differentiation into matrix-secreting osteoblasts. Osteogenesis occurs not only during embryogenesis but also during growth, after an injury, and in normal homeostatic maintenance. While much is known about osteogenesis-associated regulatory genes, the role of microRNAs (miRNAs), which are epigenetic regulators of protein expression, is just beginning to be explored. While miRNAs do not abrogate all protein expression, their purpose is to finely tune it, allowing for a timely and temporary protein down-regulation.Recent findingsThe last decade has unveiled a multitude of miRNAs that regulate key proteins within the osteogenic lineage, thus qualifying them as "ostemiRs." These miRNAs may endogenously target an activator or inhibitor of differentiation, and depending on the target, may either lead to the prolongation of a progenitor maintenance state or to early differentiation. Interestingly, cellular identity seems intimately coupled to the expression of miRNAs, which participate in the suppression of previous and subsequent differentiation steps. In such cases where key osteogenic proteins were identified as direct targets of miRNAs in non-bone cell types, or through bioinformatic prediction, future research illuminating the activity of these miRNAs during osteogenesis will be extremely valuable. Many bone-related diseases involve the dysregulation of transcription factors or other proteins found within osteoblasts and their progenitors, and the dysregulation of miRNAs, which target such factors, may play a pivotal role in disease etiology, or even as a possible therapy

Morphogenetic and regulatory mechanisms during developmental chondrogenesis: New paradigms for cartilage tissue engineering

Cartilage is the first skeletal tissue to be formed during embryogenesis leading to the creation of all mature cartilages and bones, with the exception of the flat bones in the skull. Therefore, errors occurring during the process of chondrogenesis, the formation of cartilage, often lead to severe skeletal malformations such as dysplasias. There are hundreds of skeletal dysplasias, and the molecular genetic etiology of some remains more elusive than of others. Many efforts have aimed at understanding the morphogenetic event of chondrogenesis in normal individuals, of which the main morphogenetic and regulatory mechanisms will be reviewed here. For instance, many signaling molecules that guide chondrogenesis—for example, transforming growth factor-b, bone morphogenetic proteins, fibroblast growth factors, and Wnts, as well as transcriptional regulators such as the Sox family—have already been identified. Moreover, extracellular matrix components also play an important role in this developmental event, as evidenced by the promotion of the chondrogenic potential of chondroprogenitor cells caused by collagen II and proteoglycans like versican. The growing evidence of the elements that control chondrogenesis and the increasing number of different sources of progenitor cells will, hopefully, help to create tissue engineering platforms that could overcome many developmental or degenerative diseases associated with cartilage defects.Catalonia (Spain) (Predoctoral fellowship 2005 FI 00864)National Institutes of Health (U.S.) (Grant NIH 1-ROI-EB003805-01A1)Translational Centre for Regenerative Medicine (Award TEMAT 1098SF