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Cartilage injuries and osteoarthritis are leading causes of disability in developed countries. The regeneration of damaged articular cartilage using cell transplantation or tissue engineering holds much promise but requires the identification of an appropriate cell source with a high proliferative propensity and consistent chondrogenic capacity. Human fetal mesenchymal stem cells (MSCs) have been isolated from a range of perinatal tissues, including first-trimester bone marrow, and have demonstrated enhanced expansion and differentiation potential. However, their ability to form mature chondrocytes for use in cartilage tissue engineering has not been clearly established. Here, we compare the chondrogenic potential of human MSCs isolated from fetal and adult bone marrow and show distinct differences in their responsiveness to specific growth factors. Transforming growth factor beta 3 (TGFβ3) induced chondrogenesis in adult but not fetal MSCs. In contrast, bone morphogenetic protein 2 (BMP2) induced chondrogenesis in fetal but not adult MSCs. When fetal MSCs co-stimulated with BMP2 and TGFβ3 were used for cartilage tissue engineering, they generated tissue with type II collagen and proteoglycan content comparable to adult MSCs treated with TGFβ3 alone. Investigation of the TGFβ/BMP signaling pathway showed that TGFβ3 induced phosphorylation of SMAD3 in adult but not fetal MSCs. These findings demonstrate that the initiation of chondrogenesis is modulated by distinct signaling mechanisms in fetal and adult MSCs. This study establishes the feasibility of using fetal MSCs in cartilage repair applications and proposes their potential as an in vitro system for modeling chondrogenic differentiation and skeletal development studies.

Pluripotent embryonic stem (ES) cells have the capability to differentiate to various cell types and may represent an alternative cell source for the treatment of cartilage defects. Here, we show that differentiation of ES cells toward the chondrogenic lineage can be enhanced by altering the culture conditions. Chondrogenesis was observed in intact embryoid body (EB) cultures, as detected by an increase in mRNA levels for aggrecan and Sox9 genes. Collagen IIB mRNA, the mature chondrocyte-specific splice variant, was absent at day 5, but appeared at later time points. Dexamethasone treatment of alginate-encapsulated EB cultures did not have a strong chondrogenic effect. Nor was chondrogenesis enhanced by alginate encapsulation compared to simple plating of EBs. However, disruption of day 5 EBs and culture as a micromass or pelleted mass, significantly enhanced the expression of the cartilage marker gene collagen type II and the transcription factor Sox9 compared to all other treatments. Histological and immunohistochemical analysis of pellet cultures revealed cartilage-like tissue characterized by metachromatically stained extracellular matrix and type II collagen immunoreactivity, indicative of chondrogenesis. These findings have potentially important implications for cartilage tissue engineering, since they may enable the increase in differentiated cell numbers needed for the in vitro development of functional cartilaginous tissue suitable for implantation.

Stem cells have considerable potential to repair damaged organs and tissues. We previously showed that prenatal transplantation of human first trimester fetal blood mesenchymal stem cells (hfMSCs) in a mouse model of osteogenesis imperfecta (oim mice) led to a phenotypic improvement, with a marked decrease in fracture rate. Donor cells differentiated into mature osteoblasts, producing bone proteins and minerals, including collagen type Iα2, which is absent in nontransplanted mice. This led to modifications of the bone matrix and subsequent decrease of bone brittleness, indicating that grafted cells directly contribute to improvement of bone mechanical properties. Nevertheless, the therapeutic effect was incomplete, attributing to the limited level of engraftment in bone. In this study, we show that although migration of hfMSCs to bone and bone marrow is CXCR4-SDF1 (SDF1 is stromal-derived factor) dependent, only a small number of cells present CXCR4 on the cell surface despite high levels of internal CXCR4. Priming with SDF1, however, upregulates CXCR4 to increase the CXCR4+ cell fraction, improving chemotaxis in vitro and enhancing engraftment in vivo at least threefold in both oim and wild-type bone and bone marrow. Higher engraftment in oim bones was associated with decreased bone brittleness. This strategy represents a step to improve the therapeutic benefits of fetal cell therapy toward being curative.

In recent years, the potential of stem cell research for tissue engineering-based therapies and regenerative medicine clinical applications has become well established. In 2006, Chung pioneered the first entire organ transplant using adult stem cells and a scaffold for clinical evaluation. With this a new milestone was achieved, with seven patients with myelomeningocele receiving stem cell-derived bladder transplants resulting in substantial improvements in their quality of life. While a bladder is a relatively simple organ, the breakthrough highlights the incredible benefits that can be gained from the cross-disciplinary nature of tissue engineering and regenerative medicine (TERM) that encompasses stem cell research and stem cell bioprocessing. Unquestionably, the development of bioprocess technologies for the transfer of the current laboratory-based practice of stem cell tissue culture to the clinic as therapeutics necessitates the application of engineering principles and practices to achieve control, reproducibility, automation, validation and safety of the process and the product. The successful translation will require contributions from fundamental research (from developmental biology to the ‘omics’ technologies and advances in immunology) and from existing industrial practice (biologics), especially on automation, quality assurance and regulation. The timely development, integration and execution of various components will be critical—failures of the past (such as in the commercialization of skin equivalents) on marketing, pricing, production and advertising should not be repeated. This review aims to address the principles required for successful stem cell bioprocessing so that they can be applied deftly to clinical applications.

Human mesenchymal stem cells (MSC) from adult and fetal tissues are promising candidates for cell therapy but there is a need to identify the optimal source for bone regeneration. We have previously characterized MSC populations in first trimester fetal blood, liver, and bone marrow and we now evaluate their osteogenic differentiation potential in comparison to adult bone marrow MSC. Using quantitative real-time RT-PCR, we demonstrated that 16 osteogenic-specific genes (OC, ON, BSP, OP, Col1, PCE, Met2A, OPG, PHOS1, SORT, ALP, BMP2, CBFA1, OSX, NOG, IGFII) were expressed in both fetal and adult MSC under basal conditions and were up-regulated under osteogenic conditions both in vivo and during an in vitro 21-day time-course. However, under basal conditions, fetal MSC had higher levels of osteogenic gene expression than adult MSC. Upon osteogenic differentiation, fetal MSC produced more calcium in vitro and reached higher levels of osteogenic gene up-regulation in vivo and in vitro. Second, we observed a hierarchy within fetal samples, with fetal bone marrow MSC having greater osteogenic potential than fetal blood MSC, which in turn had greater osteogenic potential than fetal liver MSC. Finally, we found that the level of gene expression under basal conditions was positively correlated with both calcium secretion and gene expression after 21 days in osteogenic conditions. Our findings suggest that stem cell therapy for bone dysplasias such as osteogenesis imperfecta may benefit from preferentially using first trimester fetal blood or bone marrow MSC over fetal liver or adult bone marrow MSC.

The majority of methodologies for maintaining human embryonic stem cell (hESC) pluripotency require the use of human or animal feeder cell layers, the most common being murine embryonic fibroblasts. In this study, we applied a protocol aimed at maintaining hESCs in culture without exposure to animal cells or proteins. hESCs were encapsulated in 1.1% (w/v) calcium alginate hydrogels and grown in basic maintenance medium for a period of up to 260 days. Investigation of the cell aggregates formed within the hydrogels yielded no evidence of the formation of any of the three germ layers, although the hESCs retained their pluripotency and could differentiate when they were subsequently cultured in a conditioned environment. Immunohistochemistry and RT-PCR showed that the hESC aggregates expressed protein and gene markers characteristic of pluripotency including Oct-4, Nanog, SSEA-4, TRA-1-60 and TRA-1-81. At the ultrastructural level, the cells were arranged in closely packed clusters and showed no cytoplasmic organelles, suggesting an undifferentiated state. These data show that it is possible to maintain hESCs in an undifferentiated state, without passaging or embryoid body formation, and without animal contamination.

Historically the function of biomaterials has been to replace diseased, damaged and aged tissues. First generation biomaterials, including bio ceramics, were selected to be as inert as possible in order to minimize the thickness of interfacial scar tissue. Bioactive glasses provided an alternative from the 1970’s onward; second generation bioactive bonding of implants with tissues and no interfacial scar tissue. This chapter reviews the discovery that controlled release of biologically active Ca and Si ions from bioactive glasses leads to the up-regulation and activation of seven families of genes in osteoprogenitor cells that give rise to rapid bone regeneration. This finding offers the possibility of creating a new generation of gene activating bioceramics designed specially for tissue engineering and in situ regeneration of tissues.

The inherited skeletal dysplasia osteogenesis imperfecta (OI) results in multiple fractures and is currently treated empirically. We transplanted human first-trimester fetal blood mesenchymal stem cells (MSCs) into homozygous oim mice in utero. This resulted in a two-thirds reduction in long bone fractures (P < .01), with fewer fractures per mouse (median 1, range 0-2 in mice that received transplants vs median 3, range 1-5 in mice that did not receive transplants by 12 weeks, P < .01). Nearly all mice that did not receive transplants had fractures (47 [97.9%] of 48), in contrast to 17 (58.6%) of 29 4- to 12-week-old mice that received transplants (P < .01). Transplantation was associated with increased bone strength (P < .01), thickness (P < .01), and length (P < .01), and normalization/reduction of growth plate height in 4- to 12-week-old oim was reduced in mice that underwent transplantion (P < .001). More donor cells were found in bone tissues compared with other organs (P < .001), with cells clustered in areas of active bone formation and remodeling, and at sites of fracture healing. Donor cells found in the bone expressed osteoblast lineage genes, and produced the extracellular bone structural protein osteopontin. Finally, MSC transplantation decreased bone hydroxyproline content. In conclusion, intrauterine transplantation of fetal MSCs markedly reduced fracture rates and skeletal abnormalities in a mouse model of the intermediate severity type III OI, suggesting a scientific basis for MSC treatment of affected human fetuses.

Current approaches have focused on deriving ESCs differentiation into chondrocytes from a cell source of spontaneously formed intact mesoderm in EB formation, resulting in limited yield. Our study aimed at upregulating chondrogenic differentiation of murine ESCs by enhancing mesoderm formation. Specifically, culture of mESCs with conditioned medium from a human hepatocarcinoma cell line resulted in a cell population with a gene expression pattern similar to that of primitive streak/nascent mesoderm, including up-regulation of brachyury, goosecoid, nodal, and cripto. From this cell population, reducing the embryoid body formation time resulted in enhancement of chondrogenic differentiation as evidenced by larger Alcian blue-stained cartilage nodules, higher production of sulfated glycosaminoglycan matrix, the presence of well-organised type II collagen and type II collagen, aggrecan and sox-9 gene expression. In conclusion, we present here a new approach to the generation of chondrocytes from mESCs that enhances yields and, thus, could have widespread applications in cartilage tissue engineering.

There is a need for new materials that can stimulate the body's own regenerative mechanisms and heal tissues. Porous templates (scaffolds) are thought to be required for three-dimensional tissue growth. This article discusses bone regeneration and the specifications of an ideal scaffold and the materials that may be suitable. Bioactive glasses have high potential as scaffold materials as they stimulate bone cells to produce new bone, they are degradable in the body and they bond to bone. The two types of bioactive glasses, their mechanisms for bioactivity and their potential for scaffold production are reviewed. Examples of their current clinical use are highlighted.

Embryonic stem cells (ESCs) are being investigated increasingly for their potential as a cell source for tissue engineering. Antibiotics are regularly used in ESC culture media to control contamination, although they can be cytotoxic and interfere with protein synthesis. Our aim was to examine the effects of the frequently used antibiotics gentamicin and combined penicillin and streptomycin on ESC culture using differentiation of murine ESC into type II pneumocytes as a model. Antibiotics reduced the expression of the specific marker for type II pneumocytes, SPC mRNA, by up to 60%. We also identified an adverse effect on the growth rate of differentiating embryoid bodies, causing a significant ( p < 0.05) reduction of up to 40%, and an increase in population doubling time of up to 48%. No contamination was seen in any of the cultures. Our findings suggest that the routine use of antibiotics in ESC culture should be avoided as it may reduce the efficiency of the culture system.

Embryonic stem cells (ESCs) are a potential source for the cell-based therapy of a wide variety of lung diseases for which the only current treatment is transplantation. However, distal lung epithelium, like many other endodermally derived somatic cell lineages, is proving difficult to obtain from both murine and human ESCs. We have previously obtained alveolar epithelium from ESCs, although final cell yield remained extremely low. Here, we present an optimized three-step protocol for the derivation of distal lung epithelial cells from murine ESCs. This protocol incorporates (a) treatment of early differentiating embryoid bodies with activin A to enhance the specification of the endodermal germ layer, followed by (b) adherent culture in serum-free medium and (c) the final application of a commercial, lung-specific medium. As well as enhancing the specification of distal lung epithelium, this protocol was found to yield cells with a phenotype most closely resembling that of lung-committed progenitor cells present in the foregut endoderm and the early lung buds during embryonic development. This is in contrast to our previous differentiation method, which drives differentiation through to mature type II alveolar epithelial cells. The derivation of a committed lung progenitor cell type from ESCs is particularly significant for regenerative medicine because the therapeutic implantation of progenitor cells has several clear advantages over the transplantation of mature, terminally differentiated somatic cells.

Tissue engineering is an interdisciplinary field that brings together the principles of the life sciences and medicine with those of engineering. The increase in its development over the past decade has resulted from a variety of factors; advances in genomics and proteomics, the advent of new biomaterials as potential templates for tissue growth, improvements in bioreactor design, and increased understanding of healing processes. Possibly the greatest contribution has come from our increased knowledge and understanding of stem cell biology, which is paving the way for the generation of unlimited cells of specific phenotypes for incorporation into engineered tissue constructs. Thus, tissue engineering approaches for expanding and engrafting the differentiated progeny of embryonic, fetal, or adult stem cells have major potential for tissue repair and will make a major contribution to medicine in the 21st century.

Osterix (Osx) is a transcription factor required for the differentiation of preosteoblasts into fully functioning osteoblasts. However, the pattern of Osx activation during preosteoblast differentiation and maturation has not been clearly defined. Our aim was to study Osx activation during these processes in osteoblasts differentiating from murine and human embryonic stem cells (ESC). To do this, we constructed an Osx-GFP fusion protein reporter system to track Osx translocation within the cells. The distribution of Osx-GFP at representative stages of differentiation was also investigated by screening primary osteoblasts, mesenchymal stem cells, synoviocytes, and pre-adipocytes. Our experiments revealed that Osx-GFP protein was detectable in the cytoplasm of cultured, differentiated ESC 4 days after plating of enzymatically dispersed embryoid bodies. Osterix-GFP protein became translocated into the nucleus on day 7 following transfer of differentiated ESC to osteogenic medium. After 14 days of differentiation, cells showing nuclear translocation of Osx-GFP formed rudimentary bone nodules that continued to increase in number over the following weeks (through day 21). We also found that Osx translocated into the nuclei of mesenchymal stem cells (C3H10T1/2) and pre-osteoblasts (MC3T3-E1) and showed partial activation in pre-adipocytes (MC3T3-L1). These data suggest that Osx activation occurs at a very early point in the differentiation of the mesenchymal-osteoblastic lineage.

Coculture of stem/progenitor cells with mature cells or tissues can drive their differentiation toward required lineages. Thus, we hypothesized that coculture of murine embryonic stem (ES) cells with embryonic mesenchyme from distal lung promotes the differentiation of pneumocytes. Murine ES cells were differentiated to embryoid bodies (EBs) and cultured for 5 or 12 days with pulmonary mesenchyme from embryonic day 11.5 or 13.5 murine embryos, in direct contact or separated by a membrane. Controls included EBs cultured alone or with embryonic gut mesenchyme. Histology revealed epithelium-lined channels in directly cocultured EBs, whereas EBs grown alone showed little structural organization. The lining cells expressed cytokeratin and thyroid transcription factor 1, an early developmental marker in pulmonary epithelium. Differentiation of type II pneumocytes specifically was demonstrated by the presence of surfactant protein C (SP-C) in some of the epithelial cells. None of these markers was seen in EBs cultured alone or with embryonic gut mesenchyme. Indirect coculture of EBs with lung mesenchyme resulted in a 14-fold increase in SP-C gene expression. Thus, provision of an appropriate microenvironment, in the form of pulmonary mesenchyme, appears to promote the differentiation of ES cells toward lung epithelium. Our findings may have applications in regenerative medicine strategies and the engineering of lung tissue.

Various means have been used to encourage the differentiation of embryonic stem cells (ESCs) toward specific lineages, including growth factor administration, genetic modification, and coculture with relevant cells/tissues. Cell extract-based reprogramming has recently been used to derive mature cells from nonrelated phenotypes. In this communication, we tested whether this in vitro reprogramming approach can be used to direct ESC differentiation. Permeabilized murine ESCs exposed to extracts of murine type II pneumocytes showed increased expression of surfactant protein C and its corresponding mRNA, reflecting enhanced differentiation of pneumocytes. Subsequent differentiation to a type I phenotype was demonstrated by expression of aquaporin 5. Pneumocyte formation occurred quicker than with growth factor-induced differentiation. Our findings establish that ESCs can be differentiated in vitro using cellular extracts. This model provides a tool for analysis of the key factors involved in the differentiation of ESCs to type II pneumocytes.

Understanding the biochemical and biophysical properties of live cells is fundamental for unravelling the secrets of many diseases and developing new therapies. Raman micro-spectroscopy is a powerful non-invasive technique that allows in vitro studies of individual living cells or groups of cells without the use of any labels or contrast enhancing chemicals. We describe the use of various multivariate statistical methods, such as Principal Component Analysis (PCA), Linear Discriminant Analysis (LDA) and Classical Least Square (CLS) fitting, to extract biochemical information related to various cellular events. Such methods are required because of the high complexity of the Raman spectra obtained from living cells. PCA and LDA are used to discriminate between healthy and tumor cells. A leave-one-out cross-validation method indicated high prediction accuracy (95%) in identification of tumorogenic bone cells. The CLS fitting method using commercially available biopolymers makes it possible to monitor biochemical changes during the differentiation of embryonic stem cells and foetal bone cells. The results suggest that in both cases differentiated cells are characterised by lower concentrations of RNA compared to undifferentiated cells. These studies suggest that Raman micro-spectroscopy could become an invaluable tool for in vitro cellular biochemistry studies.

Septal cartilage is widely used for the repair of soft tissue defects in the head, neck and nose. Tissue Engineering techniques are being investigated to create cartilage in vitro by seeding appropriate cells on resorbable scaffolds. In this study, human chondrocytes were cultured on macroporous bioactive glass foam scaffolds. The aim was to investigate how Raman spectroscopy could be used as a non-invasive technique to monitor the response of chondrocytes to a 3D scaffold in real time. The spectra were compared to scanning electron microscope (SEM) micrographs and immunohistochemistry results.

The Classical Least Square (CLS) fitting method was used to analyze the Raman spectra of living cells with the aim of identification of new phenotype-specific spectral markers for osteoblasts. The following chemicals were used for the CLS model: DNA, RNA, serum albumin, chymotrypsin and phosphatidyl choline. In this study we analyzed primary mature osteoblasts as well as two other cell types used as potential sources of osteoblasts: embryonic stem cells and fetal bone cells. The results obtained suggest that the Raman spectra of the cell types can be well approximated with a linear combination of the Raman spectra of the biopolymers used in the CLS model. The relative concentrations of the CLS components varied significantly between cell types, indicating that this analytical method could be used for phenotypic identification of osteoblasts.

Bioactive glasses dissolve upon immersion in culture medium, and release their constitutive ions into solution. There has been some evidence suggesting that these ionic-dissolution products influence osteoblast-specific processes. Here, the effect of 58S sol–gel-derived bioactive glass (60% SiO2, 36% CaO, 4% P2O5, in molar percentage) on primary osteoblasts derived from human fetal long bone explant cultures is investigated, and it is hypothesized that critical concentrations of sol–gel-dissolution products (consisting of a combination of simple inorganic ions) can enhance osteoblast phenotype in vitro by affecting the expression of a number of genes associated with the differentiation and extracellular matrix deposition processes. Cells were exposed to a range of 58S dosages continuously for a period of 4–14 days in monolayer cultures. Quantitative real-time RT-PCR analysis of a panel of osteoblast-specific markers showed a varied gene expression pattern in response to the material. The highest concentration of Ca and Si tested (96 and 50 ppm, respectively) promoted upregulation of gene expression for most markers (including alkaline phosphatase, osteocalcin, and osteopontin) at the latest time point, compared to non-58S-treated control, although this observation was not statistically significant. The same 58S concentration produced higher ALP activity levels and increased proliferation throughout the culture period, compared to lower dosages tested; however, the results generated were again not statistically significant. The data overall suggest that no significant effect can be ascribed to the ionic products of 58S bioactive gel-glass dissolution tested here and their ability to stimulate osteoblastic marker gene expression. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2005

Osterix is a transcription factor crucial for the normal development of the osteoblast. Here we have investigated whether the osteogenic differentiation of murine embryonic stem (ES) cells can be induced by overexpression of osterix. Differentiation was initiated by formation of embryoid bodies (EB) which were then dispersed and cultured in alpha-minimum essential medium supplemented with L-ascorbate phosphate and alpha-glycerophosphate for up to 21 days. osterix was found to induce expression of several osteoblast-specific markers, as confirmed by immunostaining and real-time RT-PCR. The expression of genes encoding osteocalcin and Cbfa1 was upregulated and the formation of mineralized bone nodules was significantly increased by osterix transfection. In combination with dexamethasone, bone nodule formation was further increased in osterix-transfected cells. Expression of both Sox-9 and PPAR-gamma, genes that are associated with chondrocyte and adipocyte differentiation, was initially increased in the osterix-transfected cells but was downregulated after day 7. This suggests that the process of osterix-induced differentiation of ES cells involves transition through an intermediate bi- or tripotential progenitor cell population. In conclusion, this cell differentiation strategy is useful not only for generating osteoblastic cells from ES cells, but also for investigating factors that influence this process and potentially delineating the ontogeny of the osteoblast.

Bone loss is a significant clinical problem, and treatments utilizing donated graft material are limited. To meet future demands in the healthcare industry, there has been a shift of outlook toward the use of bioactive materials for tissue regeneration. A number of in vivo and in vitro studies have highlighted the potential of the bioactive glass ceramic 45S5 Bioglass as a synthetic regenerative scaffold. The application of sol-gel processing techniques has led to the synthesis of mesoporous bioactive glasses with greater textural and compositional variety. In this study, we evaluated the effects of supplemented tissue culture medium containing up to 203 ppm silica prepared by static soaking of particles of 58S sol-gel bioactive glass (58% SiO(2), 33% CaO, 9% P(2)O(5)) on the in vitro proliferation and differentiation of murine and human primary osteoblasts. These extracts had a higher silica content than those used previously in studies of 45S5 Bioglass, because of the faster rates of ion exchange permitted by the higher surface area-to-volume ratio of mesoporous glass. We found that osteoblasts from both species increased their proliferation in response to the glass-conditioned medium. In addition, the extent to which supplemented medium could alter cell differentiation varied with time in culture. Proliferation induced by supplemented medium paralleled effects induced by treatment with basic fibroblast growth factor, a known mitogenic growth factor for osteoblasts. Bone nodule formation was also increased by exposure to the glass-conditioned medium and this effect was positively correlated with the dose of glass used to prepare the medium. Apoptosis was stimulated by glass-conditioned medium in murine osteoblasts, but inhibited in human osteoblasts. These data demonstrate the bioactive effects of dissolution products derived from sol-gel materials on primary osteoblasts and complements in vivo studies that indicate the suitability of this material as a bone graft substitute.

The in vitro differentiation of embryonic stem (ES) cells into specific phenotypes plays an important role in the development of stem cell therapy, tissue engineering and regenerative medicine. Currently, there are no biological assays able to characterise and monitor in situ and in real-time ES cells during the differentiation process. We applied Raman micro-spectroscopy to compare undifferentiated murine ES cells with murine ES cells in the differentiation process. The most significant differences between undifferentiated and differentiated ES cells (16 days differentiation via formation of embryoid body) were related to the nucleic acids. The decrease in the magnitude of RNA 813 cm −1 Raman peaks (25%) in the differentiated ES cells in comparison to undifferentiated ES cells, suggests that part of the RNA in the ES cells is used for the synthesis of specific proteins in the early stages of differentiation. In the same time, the DNA 786 cm −1 Raman peaks were lower by 50%, indicating that the differentiated cells are more in the G1 phase than S, G2 or M phases of the cell cycle. This result suggests that the proliferation rate of differentiated cells is reduced following development of a mature phenotype. This study shows the feasibility of using Raman micro-spectroscopy to monitor in situ and in real-time the differentiation of ES cells by using the intensity of Raman peak of nucleic acids as differentiation markers.

The pluripotency and high proliferative index of embryonic stem (ES) cells make them a good potential source of cells for tissue engineering purposes. We have shown that ES cells can be induced to differentiate in vitro into pulmonary epithelial cells (type II pneumocytes) using a serum-free medium designed for the maintenance of mature distal lung epithelial cells in culture (SAGM). However, the resulting cell cultures were heterogeneous. Our aim in this study was to attempt to increase pneumocyte yield and differentiation state by determining which medium components enhance the differentiation of pneumocytes and modifying the medium accordingly. Quantitative RT-PCR was used to measure changes in the expression of a type II pneumocyte-specific gene, surfactant protein C (SPC), in response to alterations in the cell culture medium. Results suggested that most individual SAGM growth factors were inhibitory for type II pneumocyte differentiation, with the largest increases in SPC expression (approximately threefold) being observed upon removal of retinoic acid and triiodothryonine. However, large standard deviations occurred between replicates, illustrating the highly variable nature of ES cell differentiation. Nevertheless, these observations represent an initial step towards achieving directed differentiation of pneumocytes from stem cells that could lead to their purification for tissue engineering purposes.

Summary Aim: To quantify the chronological sequence of changes in the morphology and immunoreactivity for neurotransmitters in the pylorus of an animal model of infantile hypertrophic pyloric stenosis and phenylketonuria. Method: Thirty specimens of pylorus from hph-1 mice and age/sex matched controls (age range: 10–180 days) were examined using conventional histology and immunohistochemistry for a variety of antigens: protein gene product 9.5, a pan neuronal marker; vasoactive intestinal polypeptide; nitric oxide synthase two antigens coalesced to the same inhibitory neurons in humans; substance P, a potent excitatory neurotransmitter; and calcitonin gene related peptide, a neurotransmitter implicated in the somatic afferent innervation of the stomach.The changes in the morphology of the muscle layers were quantified and statistically analysed for each age group (10, 20, 40, 90 and 180 days). Results: Between 10 and 90 days of age, all muscle layers of the hph-1 mice were hypertrophied, for example, 10 days, hph-1 longitudinal muscle mean diameter ¼ 3.4, control ¼ 1.8; hph-1 circular muscle width ¼ 11.5, control ¼ 4.7. The hph-1 mice were significantly smaller during this period (40 days, hph-1 weight ¼ 10 g, control ¼ 25 g).There was no change in the pattern of expression of the antigens examined within the hph-1 mice compared with the controls. Conclusion: Hph-1 mice develop a transient smooth muscle hypertrophy of the pylorus attended by gastric distension and failure to gain weight.These changes resolve as the pyloric muscle hypertrophy resolves.

We have previously shown osteogenic differentiation of mouse embryonic stem (ES) cells and temporal enrichment with osteoblastic cells, by stimulation with serum-containing culture medium supplemented with beta-glycerophosphate, ascorbate, and dexamethasone. In our present study we have used similar culture conditions to further investigate osteogenic differentiation of mouse ES cells. Using reverse transcription-polymerase chain reaction (RT-PCR) we demonstrated the expression of genes associated with osteoblast differentiation including the bone matrix protein osteocalcin and the transcription factor Cbfa-1/runx2. Furthermore, results of cDNA microarray analysis, and subsequent RT-PCR analysis of differentiating ES cells after exposure to osteogenic stimuli, revealed a combination of upregulation of genes involved in osteoblast differentiation including osteopontin, HSP-47, and IGF-II coupled with downregulation of genes involved in differentiation of other phenotypes such as the neuroectoderm factor Stra-13. Finally, we have applied magnetically activated cell-sorting methods to ES cell cultures treated with osteogenic stimuli and, using an antibody to cadherin-11, have purified a subpopulation of cells with osteoblastic characteristics.

Raman microspectroscopy was used to determine biochemical markers during the differentiation of embryonic murine stem cells (mES) in vitro. Such markers are useful to determine the differentiation status of ES cells cultured on biomaterials. Raman spectra of mES cells as undifferentiated, spontaneously differentiated (4 days), and differentiated cells via formation of embryoid bodies (16, 20 days) were analyzed. Unsupervised hierarchical cluster analysis and principal component analysis were used to determine biochemical differences between mES cells in various states of differentiation. The undifferentiated cells were characterized by high scores of the first principal component (PC1, 49% variance). Similarity between the PC1 loading and the Raman spectrum of RNA indicated a high concentration of RNA in mES cells compared to differentiated cells. The ratio between the peak areas of RNA and proteins was used as a measure of mRNA translation. Using the same peak area ratio, it was possible to differentiate even between mES as undifferentiated and in early stages of differentiation (4 days). These findings were correlated with biological studies reporting high levels of nontranslated mRNA during early embryonic development. Therefore, the RNA translation obtained from the Raman spectra can be used as marker of differentiation state of mES cells.

Historically the function of biomaterials has been to replace diseased or damaged tissues. Recent findings show that controlled release of the ionic dissolution products of bioactive glasses results in regeneration of tissues. The mechanism for in situ tissue regeneration involves upregulation of seven families of genes that control the osteoblast cell cycle, mitosis and differentiation. In the presence of critical concentrations of Si and Ca ions, within 48 h osteoblasts that are capable of differentiating into a mature osteocyte phenotype begin to proliferate and regenerate new bone. Osteoblasts that are not in the correct phase of the cell cycle and unable to proceed towards differentiation are switched into apoptosis by the ionic dissolution products. A controlled release of soluble Ca and Si from bioactive glass--resorbable polymer composites leads to vascularised soft tissue regeneration. Gene activation by controlled ion release provides the conceptual basis for molecular design of a third generation of biomaterials optimised for in situ tissue regeneration.

Tissue engineering is a multidisciplinary area of research aimed at regeneration of tissues and restoration of function of organs through implantation of cells/tissues grown outside the body, or stimulating cells to grow into implanted matrix. In this short review, some of the most recent developments in the use of stem cells for tissue repair and regeneration will be discussed. There is no doubt that stem cells derived from adult and embryonic sources hold great therapeutic potential but it is clear that there is still much research required before their use is commonplace. There is much debate over adult versus embryonic stem cells and whether both are required. It is probably too early to disregard one or other of these cell sources. With regard to embryonic stem cells, the major concern relates to the ethics of their creation and the proposed practice of therapeutic cloning.

Background. Inflammation, epithelial cell injury, and development of fibrosis and airway obliteration are the major histological features of posttransplant obliterative bronchiolitis (OB). The expression of inducible nitric oxide synthase (iNOS) in the damaged epithelium, accompanied by peroxynitrite, suggests that endogenous nitric oxide (NO) mediates the epithelial destruction preceding obliteration. To elucidate the role of NO in this cascade, heterotopic bronchial allografts were studied in pigs. Methods. Allografts or autografts were harvested serially 3-90 days after transplantation and processed for histology and immunocytochemistry for iNOS, nitrotyrosine, a marker of peroxynitrite formation, and superoxide dismutase (SOD). Results. During initial ischemic damage to the epithelium, iNOS, nitrotyrosine, and SOD were found to be strongly expressed in the epithelium of all implants as well as later, after partial recovery, parallel to onset of epithelial destruction and subsequent airway obliteration in allografts. The levels of expression of iNOS in fibroblasts during the early phase of obliteration paralleled the onset of fibrosis. Constant expression of iNOS and SOD, but not nitrotyrosine, occurred in autografts and allografts with blocked alloimmune response. Conclusions. These findings suggest that an excessive amount of NO promotes posttransplant obliterative bronchiolitis by destroying airway epithelium and stimulating fibroblast activity. SOD may provide protection by binding reactive molecules and preventing peroxynitrite formation.

We have cloned 3 novel murine cDNAs encoding proteins containing an alpha/beta hydrolase fold; a catalytic domain found in a very wide range of enzymes. These proteins belong to the prosite UPF0017 uncharacterized protein family and we have named them lung alpha/beta hydrolase 1, 2, and 3 (LABH) since they were cloned from lung cDNA. All have 9 coding exons, encoding 412, 425, and 411 residue proteins respectively (46-48 kDa); LABH1 being closely related to LABH3 having 45% identity. All 3 proteins have a single predicted amino-terminus transmembrane domain. An alignment of family members from different phyla enabled the identification of the LABH1 catalytic triad as Ser211, Asp337, and His366. mRNA expression levels of LABH1 and 3 were highest in liver and LABH2 highest in testis. These findings suggest that the LABH proteins consist of a novel family of membrane bound enzymes whose function has yet to be determined.

Age-related changes in the expression of the growth associated protein GAP-43/B-50, and the neuropeptides substance P and calcitonin gene-related peptide (CGRP) were investigated in the sensory neurones of rat dorsal root ganglia, dorsal horns of the spinal cord and subintimal knee synovium. The two time-points studied were 2 months (young adults) and 14-month (aged)-old Sprague Dawley rats. Dorsal root ganglia: In young adults, 40 and 35% of the L4-L5 dorsal root ganglion neurones were positive for GAP-43/B-50 with a 1.5 fold increase in frequency in aged rats at the L5 ganglion. GAP-43/B-50 was strongly expressed by the non-neuronal satellite cells of some medium and many large sized neurones in aged rats. There were marked reciprocal shifts between small and medium sized sensory neurones in respect to their substance P and CGRP expression profiles. Dorsal horn of the spinal cord: there was a 1.3 fold decrease of substance P at L5 level and a 1.3 and 1.5 fold decrease of CGRP at L4-L5 levels in aged rats, respectively. Synovial membrane: There was a 2.3 fold increase in GAP-43/B-50 and a 2.5 fold decrease of CGRP with no changes in substance P expression. These results indicate that (i) primary sensory neurones undergo age-related changes already in early stages of aging, (ii) aging may result in a reduction of substance P and CGRP axonal transport, and (iii) reduced numbers of CGRP containing synovial perivascular fibres may imply a deficient regulation of the synovial microvasculature and therefore metabolic homeostasis of the joint in aged subjects.

We determined the distribution of ET(A) and ET(B) receptors in pulmonary arteries from pulmonary hypertensive patients and control subjects, using in vitro autoradiography, and investigated their role in mediating the proliferative effects of endothelin-1 (ET-1) on distal human pulmonary artery smooth muscle cells (PASMCs). Distal arteries possessed more medial [(125)I]-ET-1 binding sites (105 +/- 10 versus 45 +/- 6 amol/mm(2); p < 0.001) and a greater proportion of ET(B) receptors than proximal arteries (36 +/- 3% versus 3 +/- 1%; p < 0.001). Receptor density in distal arteries and lung parenchyma was twofold greater (p < 0.05) in pulmonary hypertensive patients than in control subjects. ET-1 (10(-9)-10(-7) mol/L) stimulated DNA synthesis (147 +/- 10% of control subjects; p < 0.05) and attenuated the antiproliferative action of cicaprost and forskolin on PASMCs, these effects being mediated via ET(A) and ET(B) receptors. Serum-stimulated proliferation was attenuated by inhibiting either endogenous ET-1 release with phosphoramidon (10(-5) mol/L) or its action with PD145065 (10(-5) mol/L). Cicaprost (10(-10)-10(-7) mol/L) inhibited ET-1 release from PASMCs (49 +/- 16% of control after 24 h; p < 0.001) and increased intracellular cAMP levels, whereas ET(B) receptor stimulation selectively reduced cAMP levels. In conclusion, ET(A) and ET(B) receptors are differentially distributed in human pulmonary arteries. Both receptors promote the proliferation of PASMCs in vitro and may contribute to vascular remodeling in pulmonary hypertension.

Functional studies in gene-knockout and transgenic mice systems have shown that lymphotoxin-alpha and lymphotoxin-beta (LT-alpha and LT-beta) are of fundamental importance in peripheral lymphoid organ development, but it remains unclear what role these cytokines have to play in the adult immune response and in the pathogenesis of disease. In this study, a polyclonal anti-serum to human LT-beta was used to investigate the distribution of LT-beta by immunohistochemistry in normal and diseased tissues. In the gut, lymph nodes, spleen, and tonsil, there was some LT-beta present on a variety of lymphoid cell types. In contrast, strong staining for LT-beta was observed on plasma cells and a subpopulation of CD4+ T cells in tissues affected by chronic inflammatory disease or infection, for example in inflammatory bowel disease, and in lymph nodes obtained from patients with sarcoidosis and tuberculosis. In tuberculous and sarcoid lymph nodes, LT-beta expression also occurred on some but not all epithelioid histiocytes within granulomas and on multi-nucleated giant cells. These findings support a role for LT-beta in human disease and suggest that it might represent a therapeutic target in a variety of common infective or inflammatory disorders.

We report the first Raman spectra of individual living and dead cells (MLE-12 line) cultured on bioinert standard poly-L-lysine coated fused silica and on bioactive 45S5 Bioglass®measured at 785 nm laser excitation. At this excitation wavelength no damage was induced to the cells even after 40 minutes irradiation at 115 mW power, as indicated by cell morphology observation and trypan blue viability test. We show that shorter wavelength lasers, 488 nm and 514 nm, cannot be used because they induce damage to the cells at very low laser powers (5 mW) and short irradiation times (5–20 minutes). The most important differences between the spectra of living and dead cells are in the 1530–1700 cm−1range, where the dead cells have strong peaks at 1578 cm−1and 1607 cm−1. Other differences occur around the DNA peak at 1094 cm−1. Our study establishes the feasibility of using the 785 nm laser for anin situreal-time non-invasive method to follow biological events (proliferation, differentiation, cell death, etc.) within individual cells cultured on bioactive scaffolds in their physiologic environment over long periods of time.

To identify changes in gene expression associated with emphysema, we used differential display to compare RNA extracted from emphysematous lungs with that of unused donor tissues taken at the time of transplant. A differentially expressed sequence was identified corresponding to the 3' end of a novel human complementary DNA (cDNA) of unknown function. The human and mouse cDNA sequences were completed by 5' rapid amplification of cDNA ends. We have named it DEXI for dexamethasone-induced transcript. DEXI messenger RNA (mRNA) was upregulated 147% in emphysematous tissue compared with donor tissue. DEXI mRNA was also upregulated 230% by dexamethasone treatment of A549. The increase in expression of DEXI found in emphysema patients' tissues may be owing to their known treatment with corticosteroids. The human DEXI gene is intronless and the predicted open reading frame encodes a 95-residue acidic protein. Database searches revealed the presence of homologues only in mammals, and a human pseudogene. The protein has a predicted central transmembrane domain and a carboxy-terminal leucine zipper. The human mRNA has a single 1.3-kb transcript. We suggest that the increased expression of DEXI in emphysema may either be relevant to disease progression or be indicative of glucocorticoid responsiveness in treated patients.