Critical steps in the isolation and expansion of adipose-derived stem cells for translational therapy.

Since the discovery of adipose-derived stem cells (ASCs), there have been high expectations of their putative clinical use. Recent advances support these expectations, and it is expected that the transition from pre-clinical and clinical studies to implementation as a standard treatment modality is imminent. However ASCs must be isolated and expanded according to good manufacturing practice guidelines and a basic assurance of quality, safety, and medical effectiveness is needed for authorisation by regulatory agencies, such as European Medicines Agency and US Food and Drug Administration. In this review, a collection of studies investigating the influence of different steps of the isolation and expansion protocol on the yield and functionality of ASCs has been presented in an attempt to come up with best recommendations that ensure potential beneficial clinical outcome of using ASCs in any therapeutic setting. If the findings confirm the initial observations of beneficial effects of ASCs, the path is paved for implementing these ASC-based therapies as standard treatment options.

Increased connective tissue attachment to silicone implants by a water vapor plasma treatment.

Polydimethylsiloxane (PDMS) is the most common type of silicone polymer for the fabrication of implantable medical devices. Because of its inherent hydrophobic nature, the PDMS surface does not readily promote cellular adhesion, which leads to diverse clinical issues. Previously, we reported a simple water vapor plasma treatment of PDMS surfaces that resulted in stable long-term wettability and excellent in vitro cell compatibility. In this work, we report investigation of the in vivo local responses to PDMS implants treated by water vapor plasma using a subcutaneous rat model. The local tissue responses were assessed after 2 and 4 weeks of implantation by means of macroscopic and histomorphometric analysis. After 2 weeks of implantation, the plasma-treated implants elicited the formation of fibrous tissue capsules that were significantly thinner, more adherent, and vascularized than the control counterparts. The improved cell adhesion was correlated with an increased amount of cells attached to the implant surface after retrieval. There was no difference in the inflammatory response between untreated and treated samples. This study provides a rational approach to optimize the long-term performance of silicone implants, which is likely to have a significant impact in clinical applications demanding enhanced tissue integration of the implants.

Responses of fibroblasts and glial cells to nanostructured platinum surfaces.

The chronic performance of implantable neural prostheses is affected by the growth of encapsulation tissue onto the stimulation electrodes. Encapsulation is associated with activation of connective tissue cells at the electrode's metallic contacts, usually made of platinum. Since surface nanotopography can modulate the cellular responses to materials, the aim of the present work was to evaluate the 'in vitro' responses of connective tissue cells to platinum strictly by modulating its surface nanoroughness. Using molecular beam epitaxy combined with sputtering, we produced platinum nanostructured substrates consisting of irregularly distributed nanopyramids and investigated their effect on the proliferation, cytoskeletal organization and cellular morphology of primary fibroblasts and transformed glial cells. Cells were cultured on these substrates and their responses to surface roughness were studied. After one day in culture, the fibroblasts were more elongated and their cytoskeleton less mature when cultured on rough substrates. This effect increased as the roughness of the surface increased and was associated with reduced cell proliferation throughout the observation period (4 days). Morphological changes also occurred in glial cells, but they were triggered by a different roughness scale and did not affect cellular proliferation. In conclusion, surface nanotopography modulates the responses of fibroblasts and glial cells to platinum, which may be an important factor in optimizing the tissue response to implanted neural electrodes.

Effect of oxygen concentration, culture format and donor variability on in vitro chondrogenesis of human adipose tissue-derived stem cells.

BACKGROUND: The chondrogenic differentiation potential of the easily accessible adipose tissue-derived stem cells (ASCs) is of particular interest within the field of tissue engineering for treating cartilage defects. However, no consensus has been reached as to which oxygen tension is more beneficial for the differentiation process. MATERIALS & METHODS: In this investigation, the impact of available oxygen was investigated to identify optimal conditions for human ASC chondrogenesis in vitro. Four physiologically relevant oxygen concentrations of 15, 10, 5 and 1% were compared with ambient air condition, and the ASCs originating from six unrelated donors were subjected to chondrogenic induction in high-density pellet cultures. RESULTS: The qualitative and quantitative assessment of accumulated extracellular matrix and the gene-expression analysis revealed marked interindividual differences, nevertheless the chondrogenic process was optimally supported in high-density pellet setup at ambient or 15% oxygen concentrations, irrespective of the origin of cells. The histochemical analysis based on alcian blue staining demonstrated that the differentiation took place in a gradient-like fashion, displaying highest levels in restricted regions, most often adjacent to the periphery. The two lowest hypoxic conditions, at 5 and 1% oxygen, seemed to have an inhibitory effect. CONCLUSION: The micropellet cultures at ambient or 15% oxygen concentration provided the most suitable environment for inducing chondrogenesis in ASCs. Furthermore, in light of the fact that the induction appeared in a zone-dependent manner, this format lends itself as a suitable model for further analysis of the relationship between chondrogenic differentiation and the gradient of nutrients.

The influence of glancing angle deposited nano-rough platinum surfaces on the adsorption of fibrinogen and the proliferation of primary human fibroblasts.

We have used the glancing angle deposition (GLAD) method as a simple and fast method to generate nano-rough surfaces for protein adsorption experiments and cell assays. The surface roughness and the detailed geometrical surface morphology of the thin films were characterized by atomic force microscopy (AFM) and scanning electron microscopy (SEM). As the GLAD deposition angle approaches grazing incidence, sharp and whisker-like columnar protrusions are formed. Smaller and less sharp surface features appear for the thin films synthesized at higher deposition angles. By changing the GLAD deposition angle together with the total amount of mass deposited per area on the respective surfaces, the size of the surface features can be varied on the nanoscale. Using the GLAD topographies as model surfaces, we have investigated the influence of the nano-roughness on fibrinogen adsorption and on the proliferation of primary human fibroblasts. It is found that fibrinogen, an important blood protein, preferentially adheres on the whisker-like nano-rough substrates in comparison to a flat surface. Furthermore, the proliferation of the human fibroblasts is significantly reduced on the nano-rough substrates. These results demonstrate that the GLAD technique can be used to fabricate nano-rough surface morphologies that significantly influence both protein and cellular adhesion to surfaces and are therefore well suited for biological assays.

Transcriptional signature of human adipose tissue-derived stem cells (hASCs) preconditioned for chondrogenesis in hypoxic conditions.

Hypoxia is an important factor involved in the control of stem cells. To obtain a better insight into the phenotypical changes brought about by hypoxic preconditioning prior to chondrogenic differentiation; we have investigated growth, colony-forming and chondrogenic capacity, and global transcriptional responses of six adipose tissue-derived stem cell lines expanded at oxygen concentrations ranging from ambient to 1%. The assessment of cell proliferation and colony-forming potential revealed that the hypoxic conditions corresponding to 1% oxygen played a major role. The chondrogenic inducibility, examined by high-density pellet model, however, did not improve on hypoxic preconditioning. While the microarray analysis revealed a distinctive inter-donor variability, the exposure to 1% hypoxia superseded the biological variability and produced a specific expression profile with 2581 significantly regulated genes and substantial functional enrichment in the pathways of cell proliferation and apoptosis. Additionally, exposure to 1% oxygen resulted in upregulation of factors related to angiogenesis and cell growth. In particular, leptin (LEP), the key regulator of body weight and food intake was found to be highly upregulated. In conclusion, the results of this investigation demonstrate the significance of donor demographics and the importance of further studies into the use of regulated oxygen tension as a tool for preparation of ASCs in order to exploit their full potential.

Continuous hypoxic culturing maintains activation of Notch and allows long-term propagation of human embryonic stem cells without spontaneous differentiation.

OBJECTIVE: The maintenance of pluripotency of human embryonic stem cells (hESCs) requires a high efficiency of self-renewal. During in vitro propagation, however, hESCs have a propensity to differentiate spontaneously. In this study, we assessed the nature of hESC responses to hypoxic conditions. MATERIALS AND METHODS: Human embryonic stem cells were grown in normoxic and hypoxic conditions, and the cells expressing Oct4 and stage-specific embryonic antigen-1 were identified by indirect immunofluorescence. The transcriptional expression of Nanog, Notch1, and Oct4 was determined by a real-time reverse transcription-polymerase chain reaction, and the inhibition of Notch-mediated signalling was achieved with a gamma-secretase inhibitor. RESULTS: In contrast to culture at 21% oxygen, where the colonies displayed a marked degree of differentiation, we found that during exposure to 5% oxygen, the hESC colonies displayed a homogenous and flat morphology that was consistent with the presence of Oct4-positive phenotype, indicating no spontaneous differentiation. When cultured at 5% oxygen for either 4 weeks or up to 18 months, high levels of Nanog and Notch1 transcriptional expression were detected, albeit the expression was significantly lower during longer exposure. The suppression of differentiation was rapidly reversed on transfer of the hypoxic cultures to normoxic conditions. Looking into the molecular mechanisms of the maintenance of self-renewal at low oxygen tensions, we found that inhibition of Notch signalling fully abrogated the hypoxic induction of undifferentiated phenotype. CONCLUSION: Our data, thus, indicate that hypoxic exposure has the capacity to sustain long-term self-renewal of hESCs and that this effect is mediated through activation of Notch.

Response of chronic hypoxic cells to low dose-rate irradiation.

PURPOSE: Compare the sensitivity of human cells in vitro to low dose-rate irradiation in air and in moderate hypoxia (4% O2). MATERIALS AND METHODS: Continuous low dose-rate beta-irradiation at a dose rate of 0.015 or 0.062 Gy/h was given to human T-47D breast cancer cells by incorporation of [3H] -labelled valine into cellular protein. Acute irradiation at a dose rate of 0.4 Gy/min was performed using [137Cs]gamma-irradiation. Cells were cultivated in an atmosphere with 4% O2 using an INVIVO2 hypoxia cabinet. RESULTS: When grown in ambient air with continuous irradiation, T-47D cells were able to continue growth for at least 23 weeks at a dose-rate of 0.015 Gy/h with a surviving fraction stabilized at around 60%. When the dose rate was increased to 0.062 Gy/h the cell culture died out after about 23 days (corresponding to about 22 Gy). When grown in an atmosphere with 4% O2 we surprisingly found that the continuously irradiated T-47D cells (0.015 Gy/h) were severely inhibited in their growth, and cell death became extensive after about 3 weeks while un-irradiated cells continued growth seemingly unaffected by this low oxygenation. Peri cellular oxygenation varied between 4% and below 0.1% over an ordinary passage due to diffusion-limitations through the 2 mm deep medium. Online O2-recordings over a whole passage showed that oxygen was more depleted in the irradiated compared to the un-irradiated cultures indicating increased respiration during irradiation. While cells growing attached to the bottom were inhibited and inactivated during irradiation it was found that cells attached high up in the neck region, i.e., having only a shallow layer of medium above them, survived and formed colonies. When cells cultivated in 4% O2 for 7 weeks were irradiated with acute doses of 137Cs gamma-rays, the radiosensitivity was the same as for cells cultivated in ambient air. CONCLUSION: Continuous irradiation with 0.015 Gy/h for several weeks results in a stronger inhibition for T-47D cells grown in an atmosphere with 4% as compared to 20% O2. The data indicate that this may be due to increased oxygen consumption resulting in more severe hypoxia in [3H]-incorporating compared to control (un-irradiated) cells.

Natural killer cell-mediated basal and interferon-enhanced cytotoxicity against liver cancer cells is significantly impaired under in vivo oxygen conditions.

Current immunostimulatory treatment protocols of cancer are often met with little success. Several lines of evidence indicate that the tumour microenvironment may impair the cytotoxic activity of natural killer (NK) cells. In this study, the NK cell-mediated killing of liver-derived cells was investigated at oxygen concentrations conform to those present in the human body at physiological and pathological conditions. The in vivo-relevant oxygen concentrations corresponding to 1, 2 and 6% were compared to those of the ambient air (21%) for their effect on the lysis of four liver-derived cell lines and the highly susceptible K562 cells. Exposure to each of the hypoxic conditions had a significantly inhibitory effect on NK cytotoxicity. Treatment with interferon-alpha (IFN-alpha) in hypoxia enhanced the cytotoxic potential of the NK cells less than it enhanced the cytotoxicity at ambient oxygen conditions. In summary, the oxygen tension profoundly affects both the cytoxic activity of NK cells and their activation by IFN-alpha.

Expression of butyrate response factor 1 in HTLV-1-transformed cells and its transactivation by tax protein.

Tax oncoprotein of Human T-lymphotropic virus 1 (HTLV-1) has been proposed to dysregulate the expression of a number of cellular genes, many of which play a critical role for cell proliferation. Our initial data demonstrated that the immediate-early gene butyrate response factor 1 ( BRF1) was upregulated in HTLV-1-infected cells. The ensuing studies revealed that the effect of Tax was mediated through two transcription elements. The more proximal element, located in the vicinity of TATA box, accounted for the main Tax transactivating effect, and it appeared to be a novel transcription factor-binding site. It involved the CCTCCTC sequence (nt -59/-53, relative to transcription start site) and was dubbed BRF1 Tax-responsive site (BTRS). The cellular protein(s) recruited into the formation of DNA-protein complex at this binding site were not identified. The other element, located further upstream, was a consensus cAMP-responsive site (CRE) TGACGTCA, spanning positions -400 to -393. CRE-binding protein (CREB) was found to mediate the transactivating effect of Tax at this site. Our results present the first evidence that the Tax transactivator has a capability to modulate the expression of BRF1 and that this effect is mediated by CRE and a novel BTRS motifs.

Biological characterization of three novel variants of IFN-alpha 13 produced by human placental trophoblast.

Interferon (IFN)-alpha from the human placenta was cloned and expressed with the aim to study the antiviral, antiproliferative, and immunostimulatory activities. In the present study, we describe three previously unknown sequence variants of IFN-alpha 13 originating from the villous trophoblast. The first variant differed from IFN-alpha 13 by a Cys99Arg substitution and a 10-amino acid C-terminal deletion, which led to a severe reduction of the antiviral and antiproliferative potential. The second variant with a Glu32Tyr substitution also displayed diminished antiviral and antiproliferative properties, but to a lesser extent than the first clone. For the third variant, a Ser25Pro substitution in the N-terminal part of the protein and two substitutions in the C-terminal part of the protein, Arg126Gly and Ala140Gly, resulted in diminished antiviral but not antiproliferative properties. Regardless of the altered antiviral and antiproliferative properties, all sequence variants demonstrated natural killer (NK) cell stimulatory potentials paralleling that of prototype IFN-alpha 13. Further studies are needed to gain a better understanding of the functional significance of different IFN-alpha subtypes at the maternal-fetal interface, in particular in light of the controversial role the NK cells play in the positive outcome of pregnancy.

Quantitative gene expression profiles of human liver-derived cell lines exposed to moderate hypoxia.

AIMS: To obtain better understanding of molecular events following critical oxygen shortage in liver tissue, we performed a large-scale comparison of gene expression profiles in four human liver cell lines, Chang, Hep3B, HuH7, and HepG2. METHODS: We used Atlas cDNA expression arrays from Clontech for initial screening, and quantitative real-time RT-PCR to assess the statistical significance of observed changes. RESULTS: RT-PCR analysis confirmed significantly changed levels of 24 transcripts after 24 hours incubation in 1% O(2). Among the genes most robustly up-regulated were plasminogen activator inhibitor-1 (PAI-1), insulin-like growth factor binding protein-3, and glyceraldehyde-3-phosphate dehydrogenase. Only PAI-1 was concordantly up-regulated in all four cell lines. Conversely, most down-regulated were the stress response genes, including several heat shock proteins, yet only the expression of flap endonuclease-1 was significantly decreased in all cell lines. When comparing individual cell lines, the HepG2 cells displayed a pattern of general down-regulation (83%), followed by Hep3B with 58% of genes down-regulated. In the Chang and HuH7 cells, however, the expression of most genes, 50% and 67%, respectively, remained unchanged. CONCLUSION: These studies provide information that is of importance for improved insight into the responses of not only liver tumor cells but also normal liver tissue in the conditions where physiological oxygenation is severely impaired.

[Free nerve transplants in the reconstruction of secondary defects of the peripheral nerves of the hand]. / Pouzitie vol'ného nervového transplantátu pri rekonstrukcii sekundárnych defektov periférnych nervov ruky.

BACKGROUND: Peripheral nerve of the hand significantly participate ub the physiological functions of the hand. A defect in the area of the peripheral nerve therefore represents a problem, the solution of which is in the field of microsurgical reconstruction. The study deals with the possibility of microsurgical reconstruction by use of free nerve draft. SUBJECTIVE: Clinical observation of patients after microsurgical reconstruction of the hand peripheral nerve defect. MATERIAL AND METHODS: 34 patients after lesions of the median nerve, ulnar nerve and mutual impairment of both nerves. The evaluation according the known and spread system system of Medical Research Council, Seddon 1954. RESULTS: We have achieved very good results after isolated lesions of the median nerve, while the results in this, as well a in other groups are better in younger patients and in patients, in whom the devastation of tissue was not too great. Standard results were achieved in the group with isolated injury of the ulnar nerve and the worst results in a small group of concommitant impairment of both median and ulnar nerves. CONCLUSIONS: The results indicate that this method is unambiquously appropriate in such a complicated clinical picture. Our results are in accord with the data from other literature sources. MEANING FOR PRACTICE: Inevitability of specialized centres which would deal with this problem thus reducing the necessity of secondary reconstruction operations and naturally the reduction of financial expenditure or social support provision.

Lack of protection against vertical transmission of HIV-1 by interferons produced during pregnancy in a cohort from East African republic of Malawi.

Interferons (IFNs) associated with pregnancy were studied for their possible role in inhibition of vertical transmission of the human immunodeficiency virus type 1 (HIV-1). A study group was composed of 43 HIV-1-positive mothers, of whom 15 transmitted the virus to the offspring and 28 did not. The control group included 48 HIV-1-negative mother-infant pairs. The IFN-alpha was detected only sporadically in the maternal sera from the groups of transmitters (27%), nontransmitters (21%), and controls (19%). The average levels of IFN-alpha were low, 16.3 +/- 2.5 pg/ml, 21.4 +/- 9.9 pg/ml, and 21.3 +/- 9.4 pg/ml among the transmitters, nontransmitters, and control subjects, respectively. In the cord blood, IFN-alpha was detected only on two occasions among transmitters, and on a single occasion in the control group. IFN-beta was absent from both maternal and cord blood in the study group, and found to be present in one case in the control group simultaneously in the maternal and fetal sera. In the placentas, on the other hand, both type I and II IFNs were expressed universally in the villous trophoblast, and IFN-alpha and -beta in the stromal macrophages as well. In one case among transmitters, no IFNs were detected; nevertheless, no significant difference with respect to nontransmitters could be confirmed. Our data suggest that although the placental IFNs have an antiviral potential, they are not sufficient to suppress transmission of HIV from mother to infant.

Genetic analysis reveals ongoing HIV type 1 evolution in infected human placental trophoblast.

To provide a better understanding of the role of placenta in vertical human immunodeficiency virus (HIV) transmission, we have studied the infection of placental trophoblast in a group of 15 mother-neonate pairs. By nested PCR amplification of the C2V3 env gene region, HIV-1 has been found to infect the placenta in five cases (33%). Phylogenetic analysis of the cloned sequences showed that all recovered maternal variants were of the B subtype. Further investigation into the ancestral relationships at the nucleotide level revealed that the trophoblast sequences evolved into a quasispecies population clearly distant from that observed in the mother. As expected, the populations transmitted to the trophoblast were also found to be more homogeneous than those in the mothers when characterized on the basis of pairwise nucleotide sequence distances. With regard to the predicted biological properties, the primary amino acid structure of the V3 loop domain was consistent, with a macrophage-tropic, non-syncytium-inducing phenotype in all patients. We also attempted to determine if any of a number of selected maternal or viral factors was associated with trophoblast infection. However, none of the followed parameters, including maternal age, disease stage, antiretroviral therapy, CCR5delta32 deletion status of the infant, and viral genotype, could be associated with viral transmission. Moreover, in one pair with proven trophoblast infection, HIV was also detected in the cord blood. Taken together, our data suggest that the productive trophoblast infection by HIV-1 in vivo is a relatively frequent event that may bear direct implications for a further transplacental propagation of the virus.

Differential expression of Nur77 family members in human T-lymphotropic virus type 1-infected cells: transactivation of the TR3/nur77 gene by Tax protein.

We analyzed the differential expression and regulation of three members of the Nur77 transcription factor family by the human T-lymphotropic virus type 1 (HTLV-1) Tax protein. We have demonstrated that in both HTLV-1-infected cells and Tax-expressing JPX-9 cells, TR3/nur77 is highly expressed, whereas neither NOR-1 nor NOT expression is detectable. Transient transfection analysis further confirmed the Tax transactivation of the TR3/nur77 promoter but not the NOR-1 promoter in different cell types. Furthermore, expression of a luciferase reporter gene driven by the NGFI-B (rat homolog of TR3/Nur77) response element (NBRE) provided evidence that Tax-mediated transactivation resulted in the induction of a functional protein. Cotransfection assays with the TR3/nur77 promoter sequence or the NBRE binding motif together with a series of Tax mutants have shown that Tax-induced TR3/nur77 expression is mediated by CREB/ATF-related transcription factors.

Oxygen tension and virus replication.

An evidence is accumulating that the oxygen tension exerts significant effect on the virus replication in vitro. When the in vitro oxygen tension is maintained at an in vivo physiological level, as a rule higher yields of human viruses are seen that at conventional culturing with access of an unphysiologically high oxygen concentration in ambient air. Although not fully understood, possible explanation for this phenomenon may be provided by a lowered interferon (IFN) output and increased cell replication which is often optimal at physiological oxygen tension. Furthermore, an indirect evidence suggests that the expression of some virus receptors is affected by oxygen tension. Also, the antiviral cell-mediated immunity is likely to be found oxygen tension-dependent as both the NK and cytotoxic T cell activities towards uninfected target cells are oxygen tension-sensitive. At present, the in vitro work with viruses at physiological oxygen tensions is hampered by the fact that cells adapt in the course of several weeks to the new oxygen tension. Whether viruses may adapt to different oxygen tensions is not clear. Workbenches combining safety in manipulation with hazardous viruses and the convenience of controlled gas atmosphere during both manipulation and long-term incubation have been developed. It is suggested that the in vitro virology should ensure that the physiological oxygen tension is better mimicked in the in vivo processes. Much work is to be done to determine the molecular interactions between oxygen tension-sensitive elements of the cell and infecting viruses. Of no lesser importance are the questions regarding the role of oxygen in virus tissue tropism, the cost-benefit of virus production at different oxygen tension levels, and the potential significance of oxygen tension for delivering gene effects to the selected target tissues.

Role of the Fas/Fas ligand pathway in apoptotic cell death induced by the human T cell lymphotropic virus type I Tax transactivator.

Two distinct human diseases have been described in association with human T cell lymphotropic virus type I (HTLV-I) infection: adult T cell leukaemia and tropical spastic paraparesis/HTLV-I-associated myelopathy. Although comprehensive understanding of specific mechanisms underlying pathogenesis of either disease has not yet been achieved, the viral regulatory protein Tax is believed to play a significant role. Previous studies demonstrated the potential of Tax to transform host cells. Here, it is shown that the Tax transactivator has in addition the potential to induce T cell death by apoptosis. Using an inducible system (Jurkat cell line JPX-9), significant apoptotic cell death upon Tax expression was observed. In an attempt to detect the cellular genes mediating this effect, it was found that induction of Tax was associated with marked upregulation of the Fas ligand (FasL) gene. Tax-induced apoptosis was inhibited when the Fas/FasL pathway was interrupted by YVAD-cmk, the inhibitor of ICE-like proteases. Transient expression experiments provided additional support for the putative role of endogenous FasL in Tax-induced apoptosis. Upon cotransfection with Tax-expressing plasmid, the transcriptional activity of the FasL promoter was found to be significantly upregulated in Jurkat cells and several other cell lines, as measured by reporter gene expression. Furthermore, cotransfection using different Tax mutants demonstrated that both CREB and NF-kappaB activation domains of Tax protein were required for the transactivation to take effect.