Trine Fink

Induction of Adipocyte‐Like Phenotype in Human Mesenchymal Stem Cells by Hypoxia

Human mesenchymal stem cells (hMSCs) have the capacity to differentiate along several pathways to form bone, cartilage, tendon, muscle, and adipose tissues. The adult hMSCs reside in vivo in the bone marrow in niches where oxygen concentration is far below the ambient air, which is the most commonly encountered laboratory condition. The study reported here was designed to determine whether oxygen has a role in the differentiation of hMSCs into adipocytes. Indeed, when exposed to atmosphere containing only 1% of oxygen, the formation of adipocyte‐like phenotype with cytoplasmic lipid inclusions was observed. The effect of hypoxia on the expression of adipocyte‐specific genes was determined by real‐time reverse transcription polymerase chain reaction. Interestingly, neither of the two central regulators of adipogenesis—the transcription factors peroxisome proliferator‐activated receptor γ2 (PPAR‐γ2) and ADD1/SREBP1c—was induced. Furthermore, hypoxia did not have any effect on the transcription of early (lipoprotein lipase) or late (aP2) marker genes. By the same token, neither of the mature adipocyte‐specific genes—leptin and adipophilin—was found responsive to the treatment. High level of induction, however, was observed with the PPAR‐γ–induced angiopoietin‐related gene, PGAR. The lack of an adipocyte‐specific transcription pattern thus indicates that despite accumulation of the lipid, true adipogenic differentiation did not take place. In conclusion, hypoxia appears to exert a potent lipogenic effect independent of PPAR‐γ2 maturation pathway.

Instability of standard PCR reference genes in adipose-derived stem cells during propagation, differentiation and hypoxic exposure

BackgroundFor the accurate determination of gene expression changes during growth and differentiation studies on adipose-derived stem cells (ASCs), quantitative real-time RT-PCR has become a method of choice. The technology is very sensitive, however, without a proper selection of reference genes, to which the genes of interest are normalized, erroneous results may be obtained.ResultsIn this study, we have compared the gene expression levels of a panel of twelve widely used reference genes during hypoxic culture, osteogenic and chondrogenic differentiation, and passaging of primary human ASCs. We found that several of the commonly used reference genes including 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin were unsuitable for normalization in the conditions we tested, whereas tyrosine 3/tryptophan 5-monooxygenase activation protein (YMHAZ), TATAA-box binding protein (TBP), beta-glucuronidase (GUSB) were the most stable across all conditions.ConclusionWhen determining gene expression levels in adipose-derived stem cells, we recommend normalizing transcription levels to the geometric mean of YMHAZ, TBP and GUSB.

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.

Prolonged hypoxic culture and trypsinization increase the pro-angiogenic potential of human adipose tissue-derived stem cells

BACKGROUND AIMS Transplantation of mesenchymal stromal cells (MSC), including adipose tissue-derived stem cells (ASC), is a promising option in the treatment of vascular disease. Short-term hypoxic culture of MSC augments secretion of anti-apoptotic and angiogenic cytokines. We hypothesized that prolonged hypoxic (1% and 5% oxygen) culture and trypsinization would augment ASC expression of anti-apoptotic and angiogenic cytokines and increase the angiogenic potential of ASC-conditioned media. METHODS The effects of prolonged hypoxic culture on growth and pro-angiogenic properties were investigated using human ASC cultured at 1%, 5% and 21% oxygen. The effect of trypsinization on the expression of pro-angiogenic genes was also determined. RESULTS Trypsinization induced up-regulation of the vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF-1) genes independent of oxygen concentration. The expression of VEGF and IGF-1 was up-regulated in ASC cultured at 1% oxygen for 13 days compared with 4 days. The VEGF concentration in ASC-conditioned media was higher after prolonged hypoxic culture compared with short-term culture, while the IGF-1 and chemokine (CXC motif) ligand 12 (CXCL12) concentrations were unchanged. The VEGF receptor blocker SU5416 abolished angiogenesis in a cultured rat aortic ring model. Media from cells exposed to hypoxia increased angiogenesis, an effect that was dependent on factors other than just the VEGF concentration in the added media. CONCLUSIONS Optimization of the angiogenic potential of stem cell-based therapy in the treatment of vascular disease is important. We have demonstrated that prolonged hypoxic culture and trypsinization augment the therapeutic angiogenic potential of ASC.

Effect of growth media and serum replacements on the proliferation and differentiation of adipose-derived stem cells

BACKGROUND AIMS Studies of mesenchymal stromal cells (MSC) from BM and adipose tissue have demonstrated similar differentiation potentials along the adipo-, osteo- and chondrogenic lineages. While most clinical trials have been performed using BM-derived MSC, the focus is shifting toward the use of stem cells derived from fat tissue. The aim of the current investigation was to define optimal culture conditions that would facilitate clinical use of adipose-derived stem cells (ASC). METHODS Different types and concentrations of serum replacers and basal media were tested with respect to the optimal expansion and subsequent differentiation of primary human ASC. The effect of initial seeding density on the growth of ASC was also determined. RESULTS While several of the serum replacements proved to be clearly inferior to fetal calf serum (FCS) in promoting ASC growth, the knockout serum replacement (KOSR) had expansion properties similar to those of FCS. However, with respect to the capacity to support adipo-, osteo- and chondrogenic differentiation, KOSR proved to be less consistent than FCS. Among the media formulations, modified Eagle medium alpha supported a significantly faster cell expansion than the other basal media while still maintaining the full differentiation potential of ASC. Regarding the plating density most favorable for rapid expansion, we found that initial plating densities ranging from 100 to 200 cell/cm(2) resulted in significantly shorter doubling times than plating densities both below and above that range. CONCLUSIONS Identification of the optimal basal medium and serum replacer, together with the most favorable plating density, will facilitate cell-based and tissue-engineering applications employing ASC in pre-clinical and clinical settings.

Nanoscale topography reduces fibroblast growth, focal adhesion size and migration-related gene expression on platinum surfaces

Controlling cellular responses on biomaterial surfaces is crucial in biomedical applications such as tissue engineering and implantable prosthetics. Since cells encounter various nanoscale topographic features in their natural environment, it has been postulated that surface nanotopography may be an alternative route to fabricate biomaterials with a desirable cellular response. In this framework, we investigated the responses of primary human fibroblasts to platinum substrates with different levels of surface roughness at the nanoscale. The nanorough surfaces were fabricated by using the glancing angle deposition technique (GLAD). We found that levels of cellular responses depended on the surface roughness and the size of the nanoscale features. We showed that in response to nanotopography cells spread less and have an elongated morphology, displaying signs of actin cytoskeleton impairment and reduced formation of focal adhesion complexes. Although cell growth and adhesion were impaired on the nanorough substrates, cell viability was not affected by topography. To a minor extent our results also indicate that cell migration might be reduced on the nanorough surfaces, since a significantly lower gene expression of migration related genes were found on the roughest surfaces as compared to the flat reference. The results presented here demonstrate that surface nanotopography influences fibroblasts responses on platinum, which may be used to reduce cellular adhesion on platinum implant surfaces such as implantable neural electrodes.

Comparison of human adipose-derived stem cells and bone marrow-derived stem cells in a myocardial infarction model.

Treatment of myocardial infarction (MI) with bone marrow-derived mesenchymal stem cells and recently also adipose-derived stem cells has shown promising results. In contrast to clinical trials and their use of autologous bone marrow-derived cells from the ischemic patient, the animal MI models are often using young donors and young, often immune-compromised, recipient animals. Our objective was to compare bone marrow-derived mesenchymal stem cells with adipose-derived stem cells from an elderly ischemic patient in the treatment of MI using a fully grown non-immune-compromised rat model. Mesenchymal stem cells were isolated from adipose tissue and bone marrow and compared with respect to surface markers and proliferative capability. To compare the regenerative potential of the two stem cell populations, male Sprague–Dawley rats were randomized to receive intramyocardial injections of adipose-derived stem cells, bone marrow-derived mesenchymal stem cells, or phosphate-buffered saline 1 week following induction of MI. After 4 weeks, left ventricular ejection fraction (LVEF) was improved in the adipose-derived stem cell group, and scar wall thickness was greater compared with the saline group. Adipose-derived as well as bone marrow-derived mesenchymal stem cells prevented left ventricular end diastolic dilation. Neither of the cell groups displayed increased angiogenesis in the myocardium compared with the saline group. Adipose-derived stem cells from a human ischemic patient preserved cardiac function following MI, whereas this could not be demonstrated for bone marrow-derived mesenchymal stem cells, with only adipose-derived stem cells leading to an improvement in LVEF. Neither of the stem cell types induced myocardial angiogenesis, raising the question whether donor age and health have an effect on the efficacy of stem cells used in the treatment of MI.

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.

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.

Comparative analysis of highly defined proteases for the isolation of adipose tissue-derived stem cells

BACKGROUND Before the potential of adipose tissue-derived stem cells can fully be exploited for a broad scope of tissue-engineering and cell-based therapeutical applications, an effective and reproducible method for isolation is needed. AIM To comparatively analyze five highly defined protease formulations, Blendzyme 1-4, liberase H1 and a crude collagenase mixture in the course of digestion that consisted of three 1-h intervals. METHODS The resulting digests of human adipose tissue aspirates were evaluated for the yield of nucleated cells, viability and frequency of specific lineages, in particular CD90, CD34 and CD45, by flow cytometry. The functionality of the cells was assessed as to the colony-forming capacity in limiting dilution assays. RESULTS Based on all evaluation criteria, Blendzymes 1 and 2 and liberase H1 demonstrated a superior performance and highest consistency. Blendzyme 3 clearly underperformed compared with all other enzymes, and the performance of the rest of enzymes appeared erratic. As for the length of digestion, a 2-h interval appeared optimal when weighing both the yield and functionality of the cells in the stromal vascular fractions obtained from different adipose tissue samples. CONCLUSION Our results demonstrate that the highly purified proteases provide a valuable alternative to crude collagenase preparations, especially in scenarios where a high definition and reproducibility of the digestion process is of importance.