Use of human embryonic stem cells and umbilical cord blood stem cells for research and therapy: a prospective survey among health care professionals and patients in Switzerland

BACKGROUND: Scientific progress in the biology of hematopoietic stem cells (HSCs) provides opportunities for advances in therapy for different diseases. While stem cell sources such as umbilical cord blood (UCB) are unproblematic, other sources such as human embryonic stem cells (hESCs) raise ethical concerns. STUDY DESIGN AND METHODS: In a prospective survey we established the ethical acceptability of collection, research, and therapy with UCB HSCs versus hESCs among health care professionals, pregnant women, patients undergoing in vitro fertilization therapy, parents, and HSC donors and recipients in Switzerland. RESULTS: There was overall agreement about an ethical justification for the collection of UCB for research and therapy in the majority of participants (82%). In contrast, research and therapy with hESCs was acceptable only by a minority (38% of all responders). The collection of hESCs solely created for HSC collection purposes met overall with the lowest approval rates. Hematologists displayed among the participants the highest acceptance rates for the use of hESCs with 55% for collection, 63% for research, and 73% for therapy. CONCLUSIONS: This is the first study assessing the perception of hESCs for research and therapy in comparison with UCB HSCs in different target groups that are exposed directly, indirectly, or not at all to stem cell-based medicine. Our study shows that the debate over the legitimacy of embryo-destructive transplantation medicine is far from over as particularly hESC research continues to present an ethical problem to an overwhelming majority among laypersons and even among health care professionals.

Micromass co-culture of human articular chondrocytes and human bone marrow mesenchymal stem cells to investigate stable neocartilage tissue formation in vitro

Cell therapies for articular cartilage defects rely on expanded chondrocytes. Mesenchymal stem cells (MSC) represent an alternative cell source should their hypertrophic differentiation pathway be prevented. Possible cellular instruction between human articular chondrocytes (HAC) and human bone marrow MSC was investigated in micromass pellets. HAC and MSC were mixed in different percentages or incubated individually in pellets for 3 or 6 weeks with and without TGF-beta1 and dexamethasone (±T±D) as chondrogenic factors. Collagen II, collagen X and S100 protein expression were assessed using immunohistochemistry. Proteoglycan synthesis was evaluated applying the Bern score and quantified using dimethylmethylene blue dye binding assay. Alkaline phosphatase activity (ALP) was detected on cryosections and soluble ALP measured in pellet supernatants. HAC alone generated hyaline-like discs, while MSC formed spheroid pellets in ±T±D. Co-cultured pellets changed from disc to spheroid shape with decreasing number of HAC, and displayed random cell distribution. In -T-D, HAC expressed S100, produced GAG and collagen II, and formed lacunae, while MSC did not produce any cartilage-specific proteins. Based on GAG, collagen type II and S100 expression chondrogenic differentiation occurred in -T-D MSC co-cultures. However, quantitative experimental GAG and DNA values did not differ from predicted values, suggesting only HAC contribution to GAG production. MSC produced cartilage-specific matrix only in +T+D but underwent hypertrophy in all pellet cultures. In summary, influence of HAC on MSC was restricted to early signs of neochondrogenesis. However, MSC did not contribute to the proteoglycan deposition, and HAC could not prevent hypertrophy of MSC induced by chondrogenic stimuli.

68Ga-DOTATOC PET/CT and somatostatin receptor (sst1-sst5) expression in normal human tissue: correlation of sst2 mRNA and SUVmax

By targeting somatostatin receptors (sst) radiopeptides have been established for both diagnosis and therapy. For physiologically normal human tissues the study provides a normative database of maximum standardized uptake value (SUV(max)) and sst mRNA.

Cell-specific expression of human HGF by alveolar type II cells induces remodeling of septal wall tissue in the lung: a morphometric study

Hepatocyte growth factor (HGF) is involved in development and regeneration of the lungs. Human HGF, which was expressed specifically by alveolar epithelial type II cells after gene transfer, attenuated the bleomycin-induced pulmonary fibrosis in an animal model. As there are also regions that appear morphologically unaffected in fibrosis, the effects of this gene transfer to normal lungs is of interest. In vitro studies showed that HGF inhibits the formation of the basal lamina by cultured alveolar epithelial cells. Thus we hypothesized that, in the healthy lung, cell-specific expression of HGF induces a remodeling within septal walls. Electroporation of a plasmid of human HGF gene controlled by the surfactant protein C promoter was applied for targeted gene transfer. Using design-based stereology at light and electron microscopic level, structural alterations were analyzed and compared with a control group. HGF gene transfer increased the volume of distal air spaces, as well as the surface area of the alveolar epithelium. The volume of septal walls, as well as the number of alveoli, was unchanged. Volumes per lung of collagen and elastic fibers were unaltered, but a marked reduction of the volume of residual extracellular matrix (all components other than collagen and elastic fibers) and interstitial cells was found. A correlation between the volumes of residual extracellular matrix and distal air spaces, as well as total surface area of alveolar epithelium, could be established. Cell-specific expression of HGF leads to a remodeling of the connective tissue within the septal walls in the healthy lung, which is associated with more pronounced stretching of distal air spaces at a given hydrostatic pressure during instillation fixation.

Modulation of human osteoblasts by metal surface chemistry

The use of metal implants in dental and orthopedic surgery is continuously expanding and highly successful. While today longevity and load-bearing capacity of the implants fulfill the expectations of the patients, acceleration of osseointegration would be of particular benefit to shorten the period of convalescence. To further clarify the options to accelerate the kinetics of osseointegration, within this study, the osteogenic properties of structurally identical surfaces with different metal coatings were investigated. To assess the development and function of primary human osteoblasts on metal surfaces, cell viability, differentiation, and gene expression were determined. Titanium surfaces were used as positive, and surfaces coated with gold were used as negative controls. Little differences in the cellular parameters tested for were found when the cells were grown on titanium discs sputter coated with titanium, zirconium, niobium, tantalum, gold, and chromium. Cell number, activity of cell layer-associated alkaline phosphatase (ALP), and levels of transcripts encoding COL1A1 and BGLAP did not vary significantly in dependence of the surface chemistry. Treatment of the cell cultures with 1,25(OH)2 D3 /Dex, however, significantly increased ALP activity and BGLAP messenger RNA levels. The data demonstrate that the metal layer coated onto the titanium discs exerted little modulatory effects on cell behavior. It is suggested that the microenvironment regulated by the peri-implant tissues is more effective in regulating the tissue response than is the material of the implant itself.

Cryopreservation and autotransplantation of human ovarian tissue prior to cytotoxic therapy--a technique in its infancy but already successful in fertility preservation

Increasing survival rates in young cancer patients, new reproductive techniques and the growing interest in quality of life after gonadotoxic cancer therapies have placed fertility preservation as an important issue to oncologists, fertility specialists and patients. Several techniques are now available for fertility preservation in these patients. A new promising method is cryopreservation and transplantation of ovarian cortex. Ovarian tissue can be extracted by laparoscopy without any significant delay of gonadotoxic therapy. The tissue can be cryopreserved by specialised centres of reproductive medicine and transplanted in case the women experience premature ovarian failure (POF). This review summarises the European expertise on cryopreservation and transplantation of ovarian tissue, following around 30 reported transplantations globally, resulting in six live births and several ongoing pregnancies. It emphasises that fertility preservation by the cryopreservation of ovarian tissue is a new but already a successful clinical option, which can be considered for selected cancer patients.

Tolerance of human placental tissue to severe hypoxia and its relevance for dual ex vivo perfusion

In the dual ex vivo perfusion of an isolated human placental cotyledon it takes on average 20-30 min to set up stable perfusion circuits for the maternal and fetal vascular compartments. In vivo placental tissue of all species maintains a highly active metabolism and it continues to puzzle investigators how this tissue can survive 30 min of ischemia with more or less complete anoxia following expulsion of the organ from the uterus and do so without severe damage. There seem to be parallels between "depressed metabolism" seen in the fetus and the immature neonate in the peripartum period and survival strategies described in mammals with increased tolerance of severe hypoxia like hibernators in the state of torpor or deep sea diving turtles. Increased tolerance of hypoxia in both is explained by "partial metabolic arrest" in the sense of a temporary suspension of Kleiber's rule. Furthermore the fetus can react to major changes in surrounding oxygen tension by decreasing or increasing the rate of specific basal metabolism, providing protection against severe hypoxia as well as oxidative stress. There is some evidence that adaptive mechanisms allowing increased tolerance of severe hypoxia in the fetus or immature neonate can also be found in placental tissue, of which at least the villous portion is of fetal origin. A better understanding of the molecular details of reprogramming of fetal and placental tissues in late pregnancy may be of clinical relevance for an improved risk assessment of the individual fetus during the critical transition from intrauterine life to the outside and for the development of potential prophylactic measures against severe ante- or intrapartum hypoxia. Responses of the tissue to reperfusion deserve intensive study, since they may provide a rational basis for preventive measures against reperfusion injury and related oxidative stress. Modification of the handling of placental tissue during postpartum ischemia, and adaptation of the artificial reperfusion, may lead to an improvement of the ex vivo perfusion technique.

Evaluation of early tissue reactions after lumbar intertransverse process fusion using CT in a rabbit

OBJECTIVE: The objective of the study was to evaluate tissue reactions such as bone genesis, cartilage genesis and graft materials in the early phase of lumbar intertransverse process fusion in a rabbit model using computed tomography (CT) imaging with CT intensity (Hounsfield units) measurement, and to compare these data with histological results. MATERIALS AND METHODS: Lumbar intertransverse process fusion was performed on 18 rabbits. Four graft materials were used: autograft bone (n = 3); collagen membrane soaked with recombinant human bone morphogenetic protein-2 (rhBMP-2) (n = 5); granular calcium phosphate (n = 5); and granular calcium phosphate coated with rhBMP-2 (n = 5). All rabbits were euthanized 3 weeks post-operatively and lumbar spines were removed for CT imaging and histological examination. RESULTS: Computed tomography imaging demonstrated that each fusion mass component had the appropriate CT intensity range. CT also showed the different distributions and intensities of bone genesis in the fusion masses between the groups. Each component of tissue reactions was identified successfully on CT images using the CT intensity difference. Using CT color mapping, these observations could be easily visualized, and the results correlated well with histological findings. CONCLUSIONS: The use of CT intensity is an effective approach for observing and comparing early tissue reactions such as newly synthesized bone, newly synthesized cartilage, and graft materials after lumbar intertransverse process fusion in a rabbit model.

Multimodal approach in the use of clinical scoring, morphological MRI and biochemical T2-mapping and diffusion-weighted imaging in their ability to assess differences between cartilage repair tissue after microfracture therapy and matrix-associated autologous chondrocyte transplantation: a pilot study

OBJECTIVE: The aim of the present pilot study is to show initial results of a multimodal approach using clinical scoring, morphological magnetic resonance imaging (MRI) and biochemical T2-relaxation and diffusion-weighted imaging (DWI) in their ability to assess differences between cartilage repair tissue after microfracture therapy (MFX) and matrix-associated autologous chondrocyte transplantation (MACT). METHOD: Twenty patients were cross-sectionally evaluated at different post-operative intervals from 12 to 63 months after MFX and 12-59 months after MACT. The two groups were matched by age (MFX: 36.0+/-10.4 years; MACT: 35.1+/-7.7 years) and post-operative interval (MFX: 32.6+/-16.7 months; MACT: 31.7+/-18.3 months). After clinical evaluation using the Lysholm score, 3T-MRI was performed obtaining the MR observation of cartilage repair tissue (MOCART) score as well as T2-mapping and DWI for multi-parametric MRI. Quantitative T2-relaxation was achieved using a multi-echo spin-echo sequence; semi-quantitative diffusion-quotient (signal intensity without diffusion-weighting divided by signal intensity with diffusion weighting) was prepared by a partially balanced, steady-state gradient-echo pulse sequence. RESULTS: No differences in Lysholm (P=0.420) or MOCART (P=0.209) score were observed between MFX and MACT. T2-mapping showed lower T2 values after MFX compared to MACT (P=0.039). DWI distinguished between healthy cartilage and cartilage repair tissue in both procedures (MFX: P=0.001; MACT: P=0.007). Correlations were found between the Lysholm and the MOCART score (Pearson: 0.484; P=0.031), between the Lysholm score and DWI (Pearson:-0.557; P=0.011) and a trend between the Lysholm score and T2 (Person: 0.304; P=0.193). CONCLUSION: Using T2-mapping and DWI, additional information could be gained compared to clinical scoring or morphological MRI. In combination clinical, MR-morphological and MR-biochemical parameters can be seen as a promising multimodal tool in the follow-up of cartilage repair.

In vitro characterization of immune-related properties of human fetal bone cells for potential tissue engineering applications

We describe herein some immunological properties of human fetal bone cells recently tested for bone tissue-engineering applications. Adult mesenchymal stem cells (MSCs) and osteoblasts were included in the study for comparison. Surface markers involved in bone metabolism and immune recognition were analyzed using flow cytometry before and after differentiation or treatment with cytokines. Immunomodulatory properties were studied on activated peripheral blood mononuclear cells (PBMCs). The immuno-profile of fetal bone cells was further investigated at the gene expression level. Fetal bone cells and adult MSCs were positive for Stro-1, alkaline phosphatase, CD10, CD44, CD54, and beta2-microglobulin, but human leukocyte antigen (HLA)-I and CD80 were less present than on adult osteoblasts. All cells were negative for HLA-II. Treatment with recombinant human interferon gamma increased the presence of HLA-I in adult cells much more than in fetal cells. In the presence of activated PBMCs, fetal cells had antiproliferative effects, although with patterns not always comparable with those of adult MSCs and osteoblasts. Because of the immunological profile, and with their more-differentiated phenotype than of stem cells, fetal bone cells present an interesting potential for allogeneic cell source in tissue-engineering applications.

Retaining perivascular tissue of human saphenous vein grafts protects against surgical and distension-induced damage and preserves endothelial nitric oxide synthase and nitric oxide synthase activity

OBJECTIVE: Conventional harvesting of saphenous vein used for coronary artery bypass surgery induces a vasospasm that is overcome by high-pressure distension. Saphenous vein harvested with its cushion of perivascular tissue by a "no touch" technique does not undergo vasospasm and distension is not required, leading to an improved graft patency. The aim of this study is to investigate the effect of surgical damage and high-pressure distension on endothelial integrity and endothelial nitric oxide synthase expression and activity in saphenous vein harvested with and without perivascular tissue. METHODS: Saphenous veins from patients (n = 26) undergoing coronary artery bypass surgery were prepared with and without perivascular tissue. We analyzed the effect of 300 mm Hg distension on morphology and endothelial nitric oxide synthase/nitric oxide synthase activity using a combination of immunohistochemistry, Western blot analysis, reverse transcriptase polymerase chain reaction, and enzyme assay in distended (with and without perivascular tissue) compared with nondistended (with and without perivascular tissue) segments. RESULTS: Distension induced substantial damage to the luminal endothelium (assessed by CD31 staining) and vessel wall. Endothelial nitric oxide synthase expression and activity were significantly reduced by high-pressure distension and removal of, or damage to, perivascular tissue. The effect of distension was significantly less for those with perivascular tissue than for those without perivascular tissue in most cases. CONCLUSION: The success of the saphenous vein used as a bypass graft is affected by surgical trauma and distension. Veins removed with minimal damage exhibit increased patency rates. We show that retention of perivascular tissue on saphenous vein prepared for coronary artery bypass surgery by the "no touch" technique protects against distension-induced damage, preserves vessel morphology, and maintains endothelial nitric oxide synthase/nitric oxide synthase activity.