13 - Stem cell technology for in vitro bone tissue engineering

We explain a protocol for straightforward isolation and culture of mesenchymal stem cells (MSCs) from mouse bone marrow (BM) to supply researchers with a method that can be applied in cell biology and tissue engineering with minimal requirements. Our protocol is mainly on the basis of the frequent medium change in primary culture and diminishing the trypsinization time. Mouse mesenchymal stem cells are generally isolated from an aspirate of BM harvested from the tibia and femoral marrow compartments, then cultured in a medium with Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) for 3 h in a 37 degrees C-5% CO(2) incubator. Nonadherent cells are removed carefully after 3 h and fresh medium is replaced. When primary cultures become almost confluent, the culture is treated with 0.5 ml of 0.25% trypsin containing 0.02% ethylenediaminetetraacetic acid for 2 min at room temperature (25 degrees C). A purified population of MSCs can be obtained 3 weeks after the initiation of culture.

Transmission electron microscopy reveals ultrastructural details of cells, and it is a valuable method for studying cell organelles. That is why we used this method for detailed morphological description of different adult tissue-derived stem cells, focusing on the morphological signs of their functions (proteosynthetic activity, exchange with external environment, etc.) and their comparison. Preparing a specimen from the cell culture suitable for transmission electron microscopy is, however, much more challenging than routine tissue processing for normal histological examination. There are several issues that need to be solved while working with cell pellets instead of solid tissue. Here we describe a simple protocol for the isolation and culture of mesenchymal stem cells from different adult tissues, with applications to stem cell biology and regenerative medicine. Since we are working with population of cells that was obtained after many days of passaging, very efficient and gentle procedures are highly necessary. We demonstrated that our semi-conservative approach regarding to histological techniques and processing of cells for transmission electron microscopy is a well reproducible procedure which results in quality pictures and images of cell populations with minimum distortions and artifacts. We also commented about riskiest steps and histochemical issues (e.g., precise pH, temperature) while preparing the specimen. We bring full and detailed procedures of fixation, post-fixation, infiltration, embedding, polymerization and contrasting of cell obtained from in vitro cell and tissue cultures, with modifications according to our experience. All this steps are essential for us to know more about adult stem cells derived from different sources or about other random cell populations. The knowledge about detailed ultra-structure of adult stem cells cultured in vitro are also essential for their using in regenerative medicine and tissue engineering.

White adipose tissue was long perceived as a passive lipid storage depot but it is now considered as an active and important endocrine organ. It also harbours not only adipocytes and vascular cells but also a wide array of immunologically active cells, including macrophages and lymphocytes, which may induce obesity-related inflammation. Recently, adipose tissue has been reported as a source of adult mesenchymal stem cells with wide use in regenerative medicine and tissue engineering. Their relatively non-complicated procurement and collection (often performed as liposuction during aesthetic surgery) and grand plasticity support this idea even more. We focused our research on exploring the issues of isolation and long-term cultivation of mesenchymal stem cells obtained from adipose tissue. Ultra-structural morphology of the cells cultivated in vitro has been studied and analysed in several cultivation time periods and following serial passages--up to 30 passages. In the first passages they had ultra-structural characteristics of cells with high proteosynthetic activity. Within the cytoplasm, big number of small lipid droplets and between them, sparsely placed, small and inconspicuous, electron-dense, lamellar bodies, which resembled myelin figures were observed. The cells from the later passages contained high number of lamellar electron-dense structures, which filled out almost the entire cytoplasm. In between, mitochondria were often found. These bodies were sometimes small and resembled myelin figures, but several of them reached huge dimensions (more than 1 µm) and their lamellar structure was not distinguishable. We did not have an answer to the question about their function, but they probably represented the evidence of active metabolism of lipids present in the cytoplasm of these cells or represented residual bodies, which arise after the breakdown of cellular organelles, notably mitochondria during long-term cultivation.

The aim of this study was to isolate human mesenchymal stem cells (MSCs) from the gingiva (GMSCs) and confirm their multiple differentiation potentials, including the odontogenic lineage. GMSCs, periodontal ligament stem cells (PDLSCs) and dermal stem cells (DSCs) cultures were analyzed for cell shape, cell cycle, colony-forming unit-fibroblast (CFU-F) and stem cell markers. Cells were then induced for osteogenic and adipogenic differentiation and analyzed for differentiation markers (alkaline phosphatase (ALP) activity, mineralization nodule formation and Runx2, ALP, osteocalcin (OCN) and collagen I expressions for the osteogenic differentiation, and lipid vacuole formation and PPARγ-2 expression for the adipogenic differentiation). Besides, the odontogenic differentiation potential of GMSCs induced with embryonic tooth germ cell-conditioned medium (ETGC-CM) was observed. GMSCs, PDLSCs and DSCs were all stromal origin. PDLSCs showed much higher osteogenic differentiation ability but lower adipogenic differentiation potential than DSCs. GMSCs showed the medial osteogenic and adipogenic differentiation potentials between those of PDLSCs and DSCs. GMSCs were capable of expressing the odontogenic genes after ETGC-CM induction. This study provides evidence that GMSCs can be used in tissue engineering/regeneration protocols as an approachable stem cell source.

Human Embryonic Stem Cells . Edited by J. Odorico, S. Zhang and R. Pedersen . (Pp. xxii + 391 , illustrated , ISBN 18599 62785 , £80 hardback.) Abingdon, UK : BIOS Scientific/Garland Science . It is now nearly seven years since the first description of human embryonic stem (ES) cell lines was published by Jamie Thomson. That event, closely apposed to the reports of the cloning of sheep (‘Dolly’), and then mice, by somatic nuclear transfer, catalysed substantial interest in the idea of regenerative medicine and the possibility that stem cells of all sorts might be harnessed for the replacement of tissues lost to accident or disease. At about the same time, a number of papers appeared, reporting that various somatic stem cells – which were previously thought to be committed to lineages from within their tissue of origin – might indeed exhibit substantial plasticity and be able to generate terminally differentiated cells corresponding to a much wider range of cell types. Thus, the current era of stem cell biology was born, though the concept of stem cells and their application in medicine is much older. One consequence of this resurgent interest in stem cell biology has been the publishing of several multi-author volumes devoted to stem cells and their potential. The volume Human Embryonic Stem Cells, edited by J. Odorico, S. Zhang and R. Pedersen, is the latest in a series produced by different publishing houses over the past few years. Paradoxically, however, as Jamie Thomson points out in his foreword to the present volume, progress in the field has been slow over this time. Prospects for adult stem cells have been dimmed by controversy over the phenomenon of ‘plasticity’, with the suggestion that at least some of the reported cases might be artefactual, depending upon cell fusion or rare transdifferentiation events. At the same time, progress with human ES cells was initially hindered by the difficulty of access to established lines, and the complexity of maintaining and expanding the cultures when they could be obtained. Although mouse ES cells have been available for over 20 years, they have mostly been used as tools to produce transgenic mice. With notable exceptions, few have investigated their cell biology for its own sake, and so there has been relatively little experience from studies of mouse ES cells to guide the study of their human counterparts. The development of ES cell lines was a systematic progression from the study of teratocarcinomas in the 1970s, by those who thought that these tumours might provide key insights into the mechanisms of embryonic development. It is then perhaps ironic that clues about how to control the behaviour of human ES cells in culture are now being provided by our detailed knowledge of developmental genetics, especially of the mouse. However, the human ES cell field is changing. Many laboratories in many countries have now derived human ES cell lines. In the current International Stem Cell Initiative, a collaborative venture to compare the properties of human ES cell lines derived worldwide, 75 independent lines derived in 17 laboratories and ten countries have been enrolled in the study. Meanwhile, reports are now beginning to appear in prominent journals, addressing the molecular mechanisms of human ES cell proliferation and differentiation – not merely describing their characteristics and potential for diverse differentiation. The present volume, unlike many of its competitors, focuses explicitly on human ES cells, and may be thought to mark the end of the first phase of human ES cell research. It brings together a series of authors who have contributed significantly to the field and it balances chapters discussing the basic biology of ES cells with others discussing their differentiation along specific lineages and eventual application. Despite the focus on ES cells, two early chapters provide useful comparative reviews of recent studies of adult stem cell plasticity and of mesenchymal stem cells. Further chapters address potential issues that will need to be addressed as derivatives of human ES cells are developed for eventual clinical application. Included here are two interesting and useful reviews of the ethical, legal and intellectual property aspects of human ES cell research and application. Undoubtedly, research in the new field of human ES cell biology is beginning to advance rapidly, so that it might be considered that the present volume will become rapidly obsolete. However, its well-organized and well-written chapters provide a valuable reference to the current state of the field for newcomers and established human ES cell researchers alike. Although our understanding of molecular mechanisms and the means of manipulating human ES cells is likely to change significantly in the future, much of what is contained in this volume will remain fundamental, and so of value to those working in this exciting new area of research for quite a few years to come.

Embryonic stem cells are pluripotent and capable of unlimited self-renewal. Elucidation of the underlying molecular mechanism may contribute to the advancement of cell-based regenerative medicine. In the present work, we performed a large scale analysis of the phosphoproteome in mouse embryonic stem (mES) cells. Using multiplex strategies, we detected 4581 proteins and 3970 high confidence distinct phosphosites in 1642 phosphoproteins. Notably, 22 prominent phosphorylated stem cell marker proteins with 39 novel phosphosites were identified for the first time by mass spectrometry, including phosphorylation sites in NANOG (Ser-65) and RE1 silencing transcription factor (Ser-950 and Thr-953). Quantitative profiles of NANOG peptides obtained during the differentiation of mES cells revealed that the abundance of phosphopeptides and non-phosphopeptides decreased with different trends. To our knowledge, this study presents the largest global characterization of phosphorylation in mES cells. Compared with a study of ultimately differentiated tissue cells, a bioinformatics analysis of the phosphorylation data set revealed a consistent phosphorylation motif in human and mouse ES cells. Moreover, investigations into phosphorylation conservation suggested that phosphoproteins were more conserved in the undifferentiated ES cell state than in the ultimately differentiated tissue cell state. However, the opposite conclusion was drawn from this conservation comparison with phosphosites. Overall, this work provides an overview of phosphorylation in mES cells and is a valuable resource for the future understanding of basic biology in mES cells.

Stem cell–based bone tissue engineering has been recognized as a new strategy for maxillary sinus floor elevation. More rapid bone formation may enhance this technique when simultaneous dental implant placement is desired. Adipose tissue–derived stem cells (ADSCs) and bone marrow stem cells (BMSCs) are the most well-characterized cell sources for bone regeneration, but comparative studies on the osteogenic potential of these cells have yielded conflicting conclusions. This study aimed to compare the rapid bone formation capacity of ADSCs and BMSCs in a canine sinus floor augmentation model. In in vitro studies, BMSCs had a higher proliferative ability and greater osteogenic differentiation potential at both the mRNA and protein levels. When GFP-labeled cells on calcium phosphate cement (CPC) scaffolds were implanted subcutaneously into nude mice, both ADSCs and BMSCs survived for 4 wks, but only BMSCs formed new bone. Furthermore, according to sequential fluorescence labeling results for the canine sinus, BMSCs promoted rapid and greater bone regeneration during the entire observation period. In contrast, obvious mineralization was detected starting from 3 wks after implantation in the ADSC group. These results suggest that BMSCs might be more useful than ADSCs for rapid bone regeneration for sinus augmentation with simultaneous implant placement.

Human adipose-derived mesenchymal stem (stromal) cells (hADSC) represent an attractive source of the cells for numerous therapeutic applications in regenerative medicine. These cells are also an efficient model to study biological pathways of stem cell action, tissue injury and disease. Like any other primary somatic cells in culture, industrial-scale expansion of mesenchymal stromal cells (MSC) leads to the replicative exhaustion/senescence as defined by the "Hayflick limit." The senescence is not only greatly effecting in vivo potency of the stem cell cultures but also might be the cause and the source of clinical inconsistency arising from infused cell preparations. In this light, the characterization of hADSC replicative and stressor-induced senescence phenotypes is of great interest.This chapter summarizes some of the essential protocols and assays used at our laboratories and clinic for the human fat procurement, isolation, culture, differentiation, and characterization of mesenchymal stem cells from adipose tissue and the stromal vascular fraction. Additionally, we provide manuals for characterization of hADSC senescence in a culture based on stem cells immunophenotype, proliferation rate, migration potential, and numerous other well-accepted markers of cellular senescence. Such methodological framework will be immensely helpful to design standards and surrogate measures for hADSC-based therapeutic applications.

The purpose of this study was to conduct a histomorphometric analysis of bone marrow-derived and adipose tissue-derived stem cells, associated with a xenograft block, in vertical bone constructions in rabbit calvaria. Ten rabbits received two xenograft blocks on the calvaria, after decortication of the parietal bone. The blocks were fixed with titanium screws. The blocks were combined with the bone marrow-derived mesenchymal stem cells in the bone marrow stem cell (BMSC) group (right side of the calvaria) or with the adipose tissue-derived mesenchymal stem cells in the adipose tissue stem cell (ATSC) group (left side of the calvaria). After 8 weeks, the animals were sacrificed and their parietal bones were fixed in 10% formalin for the histomorphometric analysis. The following parameters were evaluated—newly formed bone (NFB), xenogeneic residual particles (XRP), and non-mineralized tissue (NMT). The histomorphometric analysis revealed 11.9 ± 7.5% and 7.6 ± 5.6% for NFB, 22.14 ± 8.5% and 21.6 ± 8.5% for XRP, and 65.8 ± 10.4% and 70.8 ± 7.4% for NMT in groups BMSC and ATSC, respectively, with statistically significant differences in the NFB and the NMT between the groups, but no differences in the XRP. Therefore, it can be concluded that the bone marrow-derived stem cells seem to have more potential for the bone formation than do the adipose tissue-derived stem cells when used in combination with the xenogenous blocks in the vertical bone construction.

Background: Cell-based therapy has achieved good functional recovery for tissue repair. Mesenchymal stemcells (MSCs) exhibit multilineage potential, long-term viability, and capacity for self-renewal. Periosteum-derivedmesenchymal stem cells (PD cells) may be an attractive cell source for tissue engineering because of their easyaccessibility and reduced ethical concerns. Objectives: To isolate and investigate the phenotypic and functional characteristics of mesenchymal stem cellsderived from human periosteum. We also examined the differentiation of PD cells with a trilineage differentiationassay to determine whether they were MSCs.Materials and Methods: Periosteum-derived cells were cultured in osteogenic, chondrogenic or adipogenicmedia to evaluate their multilineage differentiation potential. Adherent fibroblast-like cells were analyzed byflow cytometry for MSC cell surface markers. Differentiation of PD cells into osteogenic, chondrogenic, andadipogenic lineages was also evaluated by von Kossa, Alizarin red, Alcian blue, and oil red O stains, respectively. Expression of mesenchymal stem cell markers were assessed using reverse transcriptase-polymerase chainreaction (RT-PCR) analysis. Results: We successfully isolated and expanded MSCs from human periosteum. Flow cytometry revealed thatPD cells were positive for mesenchymal adhesion cell markers (CD29, CD44, CD90, and CD105) and negative forhematopoietic markers (CD34 and CD45). In osteogenic differentiation, calcium accumulation (positive vonKossa and Alizarin red) and RUNX2, alkaline phosphatase, collagen type I, osteopontin genes were detected.In adipogenic differentiation, the cells displayed oil red O positive and expressed lipoprotein lipase andperoxisome proliferator-activated receptor-gamma (PPAR-?) associated with adipogenesis. The cells grownin chondrogenic conditions were positively stained for Alcian blue and expressed SOX-9. Conclusion: PD cells presented osteogenic, chondrogenic, and adipogenic differentiation abilities in vitro andcould provide an alternative cellular source for tissue repair in clinical applications. Keywords: Adipogenic, chondrogenic, differentiation, mesenchymal stem cells, osteogenic, periosteum

Mesenchymal stem cells have gained popularity in cell-based therapies due to their regenerative capabilities, immunomodulation properties, and paracrine activity through trophic factors. It is of utmost importance to establish clinical-grade procedures for the preparation of the mesenchymal stem cells for clinical applications. Here, we describe detailed procedures for isolation, culture, cryopreservation, and preparation of mesenchymal stem cells derived from umbilical cord as a final product under good manufacturing practices-compliant conditions.

Stabilization of hESCs in two distinct substates along the continuum of pluripotency.

A wealth of evidence attests that the organs of developing embryos, particularly the developing brain, are acutely sensitive to chemical perturbations. However, scientists know very little about how exposures to specific endogenous chemicals actually impact human development or children’s ability to learn. And there are almost no data on how the vast majority of the 84,000 chemicals currently listed in the Toxic Substances Control Act (TSCA) Inventory1—including most of the 201 compounds known to be neurotoxic to adults and the 1,000 chemicals shown to be neurotoxic to animals2—may affect developing infants. It is also unclear whether testing with animals always provides accurate insights into human developmental susceptibility. A new line of research based on human stem cells is providing important insights into how chemicals may affect neonatal development. Stem cells are the master cells capable of producing some or all of the 200-plus different types of cells in the human body. In time, some researchers believe stem cells may enable scientists to amass far more data on how exposure to environmental chemicals affects human development, particularly the development of the brain. Now is a “critical time to be talking about stem cell research in the environmental health context,” says Tracey Woodruff, director of the Program on Reproductive Health and the Environment at the University of California, San Francisco (UCSF) Medical School.

Adipose tissue-derived stem cells are of great interest because of their properties of immune modulation, tissue regeneration, and multipotent differentiation. To advance development of stem cell-based treatments, determination of the physiologic concentration of adipose tissue-derived stem cells in human adipose tissue is relevant for proper guidance of stem cell treatment dosage, oncologic safety, and evaluation of efficacy. A prospective comparative case-control study of 20 patients was conducted to determine the yield of adipose tissue-derived stem cells in periumbilical adipose tissue harvested by the widely used method of aspiration and in structurally intact adipose tissue harvested by excision. Stem cells were isolated using conventional enzymatic digestion and by a method combining enzymatic digestion with mechanical distortion. Stem cell yield was quantified by multicolor flow cytometry and colony-forming capacity. When only the conventional enzymatic digestion was used, no significant difference in adipose tissue-derived stem cell yield was observed. However, when enzymatic digestion was combined with mechanical distortion, twice as many stem cells were isolated from excised adipose tissue compared to aspirated adipose tissue. Inclusion of mechanical distortion significantly increased yield 5-fold in excised adipose tissue and 2-fold in aspirated adipose tissue. Combining enzymatic digestion and mechanical distortion, measured levels of excised adipose tissue reached 140 × 10 (95 percent CI, 62 to 220 × 10) adipose tissue-derived stem cells per gram of adipose tissue that corresponded to 26 × 10 (95 percent CI, 18 to 33 × 10) colony-forming units per gram. The study indicates that harvesting by aspiration halves the concentration of adipose tissue-derived stem cells in adipose tissue samples when compared to structural intact adipose tissue. Furthermore, the study presents stem cell yield higher than previously described in the current literature. Therapeutic, V.

Distinctive properties of stem cells are not autonomously achieved, and recent evidence points to a level of external control from the microenvironment. Here, we demonstrate that self-renewal and pluripotent properties of human embryonic stem (ES) cells depend on a dynamic interplay between human ES cells and autologously derived human ES cell fibroblast-like cells (hdFs). Human ES cells and hdFs are uniquely defined by insulin-like growth factor (IGF)- and fibroblast growth factor (FGF)-dependence. IGF 1 receptor (IGF1R) expression was exclusive to the human ES cells, whereas FGF receptor 1 (FGFR1) expression was restricted to surrounding hdFs. Blocking the IGF-II/IGF1R pathway reduced survival and clonogenicity of human ES cells, whereas inhibition of the FGF pathway indirectly caused differentiation. IGF-II is expressed by hdFs in response to FGF, and alone was sufficient in maintaining human ES cell cultures. Our study demonstrates a direct role of the IGF-II/IGF1R axis on human ES cell physiology and establishes that hdFs produced by human ES cells themselves define the stem cell niche of pluripotent human stem cells.