Marco N. Helder

Adipose tissue-derived mesenchymal stem cell yield and growth characteristics are affected by the tissue-harvesting procedure

BACKGROUND Adipose tissue contains a stromal vascular fraction that can be easily isolated and provides a rich source of adipose tissue-derived mesenchymal stem cells (ASC). These ASC are a potential source of cells for tissue engineering. We studied whether the yield and growth characteristics of ASC were affected by the type of surgical procedure used for adipose tissue harvesting, i.e. resection, tumescent liposuction and ultrasound-assisted liposuction. METHODS Frequencies of ASC in the stromal vascular fraction were assessed in limiting dilution assays. The phenotypical marker profile of ASC was determined, using flow cytometry, and growth kinetics were investigated in culture. ASC were cultured under chondrogenic and osteogenic conditions to confirm their differentiation potential. RESULTS The number of viable cells in the stromal vascular fraction was affected by neither the type of surgical procedure nor the anatomical site of the body from where the adipose tissue was harvested. After all three surgical procedures, cultured ASC did express a CD34+ CD31- CD105+ CD166+ CD45- CD90+ ASC phenotype. However, ultrasound-assisted liposuction resulted in a lower frequency of proliferating ASC, as well as a longer population doubling time of ASC, compared with resection. ASC demonstrated chondrogenic and osteogenic differentiation potential. DISCUSSION We conclude that yield and growth characteristics of ASC are affected by the type of surgical procedure used for adipose tissue harvesting. Resection and tumescent liposuction seem to be preferable above ultrasound-assisted liposuction for tissue-engineering purposes.

Effect of tissue-harvesting site on yield of stem cells derived from adipose tissue: implications for cell-based therapies

The stromal vascular fraction (SVF) of adipose tissue contains an abundant population of multipotent adipose-tissue-derived stem cells (ASCs) that possess the capacity to differentiate into cells of the mesodermal lineage in vitro. For cell-based therapies, an advantageous approach would be to harvest these SVF cells and give them back to the patient within a single surgical procedure, thereby avoiding lengthy and costly in vitro culturing steps. However, this requires SVF-isolates to contain sufficient ASCs capable of differentiating into the desired cell lineage. We have investigated whether the yield and function of ASCs are affected by the anatomical sites most frequently used for harvesting adipose tissue: the abdomen and hip/thigh region. The frequency of ASCs in the SVF of adipose tissue from the abdomen and hip/thigh region was determined in limiting dilution and colony-forming unit (CFU) assays. The capacity of these ASCs to differentiate into the chondrogenic and osteogenic pathways was investigated by quantitative real-time polymerase chain reaction and (immuno)histochemistry. A significant difference (P = 0.0009) was seen in ASC frequency but not in the absolute number of nucleated cells between adipose tissue harvested from the abdomen (5.1 ± 1.1%, mean ± SEM) and hip/thigh region (1.2 ± 0.7%). However, within the CFUs derived from both tissues, the frequency of CFUs having osteogenic differentiation potential was the same. When cultured, homogeneous cell populations were obtained with similar growth kinetics and phenotype. No differences were detected in differentiation capacity between ASCs from both tissue-harvesting sites. We conclude that the yield of ASCs, but not the total amount of nucleated cells per volume or the ASC proliferation and differentiation capacities, are dependent on the tissue-harvesting site. The abdomen seems to be preferable to the hip/thigh region for harvesting adipose tissue, in particular when considering SVF cells for stem-cell-based therapies in one-step surgical procedures for skeletal tissue engineering.

Repair, regenerative and supportive therapies of the annulus fibrosus: achievements and challenges

Lumbar discectomy is a very effective therapy for neurological decompression in patients suffering from sciatica due to hernia nuclei pulposus. However, high recurrence rates and persisting post-operative low back pain in these patients require serious attention. In the past decade, tissue engineering strategies have been developed mainly targeted to the regeneration of the nucleus pulposus (NP) of the intervertebral disc. Accompanying techniques that deal with the damaged annulus fibrous are now increasingly recognised as mandatory in order to prevent re-herniation to increase the potential of NP repair and to confine NP replacement therapies. In the current review, the requirements, achievements and challenges in this quickly emerging field of research are discussed.

Bone cell responses to high-frequency vibration stress: does the nucleus oscillate within the cytoplasm?

Mechanosensing by cells directs changes in bone mass and structure in response to the challenges of mechanical loading. Low‐amplitude, high‐frequency loading stimulates bone growth by enhancing bone formation and inhibiting disuse osteoporosis. However, how bone cells sense vibration stress is unknown. Hence, we investigated bone cell responses to vibration stress at a wide frequency range (5–100 Hz). We used NO and prostaglandin E2 (PGE2) release, and COX‐2 mRNA expression as parameters for bone cell response since these molecules regulate bone adaptation to mechanical loading. NO release positively correlated whereas PGE2 release negatively correlated to the maximum acceleration rate of the vibration stress. COX‐2 mRNA expression increased in a frequency‐dependent manner, which relates to increased NO release at high frequencies, confirming our previous results. The negatively correlated release of NO and PGE2 suggests that these signaling molecules play different roles in bone adaptation to high‐frequency loading. The maximum acceleration rate is proportional to ω3 (frequency=ω/2π), which is commensurate with the Stokes‐Einstein relation for modeling cell nucleus motion within the cytoplasm due to vibration stress. Correlations of NO and PGE2 with the maximum acceleration rate then relate to nucleus oscillations, providing a physical basis for cellular mechanosensing of high‐frequency loading.—Bacabac, R. G., Smit, T. H., Van Loon, J. J. W. A., Doulabi, B. Z., Helder, M., Klein‐Nulend, J. Bone cell responses to high‐frequency vibration stress: does the nucleus oscillate within the cytoplasm? FASEB J. 20, 858–864 (2006)

Adipose stem cells for intervertebral disc regeneration: current status and concepts for the future

•  Introduction •  Degenerative disc disease and emerging biological treatment approaches •  Stem cell sources •  Integration of ASC‐based regenerative medicine and surgery •  In vitro studies ‐  Animal models ‐  Cells in disc regeneration in vivo •  In vivo studies •  Perspective •  Conclusions

Freshly isolated stromal cells from the infrapatellar fat pad are suitable for a one-step surgical procedure to regenerate cartilage tissue

BACKGROUND AIMS Stem cell therapies are being evaluated as promising alternatives for cartilage regeneration. We investigated whether stromal vascular fraction cells (SVF) from the infrapatellar (Hoffa) fat pad are suitable for a one-step surgical procedure to treat focal cartilage defects. METHODS SVF was harvested from patients undergoing knee arthroplasty (n = 53). Colony-forming unit (CFU) assays, growth kinetics and surface marker profiles were determined, and the chondrogenic differentiation capacity of freshly isolated SVF was assessed after seeding in three-dimensional poly (L-lactic-co-epsilon-caprolactone) scaffolds. RESULTS SVF yield per fat pad varied between 0.55 and 16 x 10(6) cells. CFU frequency and population doubling time were 2.6 +/- 0.6% and +/-2 days, respectively. Surface marker profiles matched those of subcutaneous-derived adipose-derived stem cells (ASC). CFU from Hoffa SVF showed differentiation toward osteogenic and adipogenic lineages. Cartilage differentiation was confirmed by up-regulation of the cartilage genes sox9, aggrecan, collagen type II and cartilage oligomeric matrix protein (COMP), collagen II immunostaining, Alcian Blue staining and glycosaminoglycan production. Compared with passaged cells, SVF showed at least similar chondrogenic potential. CONCLUSIONS This study demonstrates that SVF cells from the infrapatellar fat pad are suitable for future application in a one-step surgical procedure to regenerate cartilage tissue. SVF shows similar favorable characteristics as cultured ASC, and chondrogenic differentiation even appears to be slightly better. However, because of variable harvesting volumes and yields, SVF from the infrapatellar fat pad might only be applicable for treatment of small focal cartilage defects, whereas for larger osteoarthritic defects subcutaneous adipose tissue depot would be preferable.

Experimental intervertebral disc degeneration induced by chondroitinase ABC in the goat

Study Design. In 2 studies, the injection of chondroitinase ABC into intervertebral discs of mature goats was evaluated as an experimental disc degeneration model. The first study analyzed the development of degeneration in time; the second study determined the optimal enzyme concentration. Objectives. To develop reproducible, slowly progressive disc degeneration in a large animal model. Summary of Background Data. Currently available, small animal models of intervertebral disc degeneration have shortcomings in the comparability to humans in terms of size, geometry, and cell population. Also, the methods to induce degeneration in the current models do not mimic human degeneration, which starts with the loss of proteoglycans. Injecting the enzyme chondroitinase ABC into the nucleus pulposus mimics the loss of proteoglycans. Methods. In Study 1, lumbar intervertebral discs of 17 goats were injected with chondroitinase ABC (0.25 U/mL) or phosphate-buffered saline. Degeneration was analyzed with radiograph analysis, MR imaging, and macroscopic and histologic scoring at 5 different time points (4, 8, 12, 18, and 26 weeks). Six control goats were analyzed. The second study used 6 goats in which 4 different concentrations of chondroitinase ABC (0.2–0.35 U/mL) or phosphate-buffered saline were injected. After 12 weeks, similar analyses as in Study 1 were performed. Results. After 12 weeks, degenerative signs were observed in all parameters in Study 1. The degeneration increased up to 18 weeks and leveled off after 26 weeks. The variability, however, was high. The second study showed a concentration dependent effect of chondroitinase ABC with all analyzed parameters. The injection of 0.25 U/mL chondroitinase ABC resulted in disc degeneration after 12 weeks without signs of severe degeneration. Conclusion. Injection of chondroitinase ABC in the caprine intervertebral disc results in mild, slowly progressive disc degeneration. This effect was optimal at a concentration of 0.25 U/mL. This is a promising model of disc degeneration that deserves further study.

Reproducible long-term disc degeneration in a large animal model

Study Design. Twelve goats were chemically degenerated and the development of the degenerative signs was followed for 26 weeks to evaluate the progression of the induced degeneration. The results were also compared with a previous study to determine the reproducibility. Objectives. The purpose of this study was determine whether this Chondroitinase ABC (CABC) induced goat model is reproducible and to study the development of the degeneration in time up to 26 weeks. Summary of Background Data. Injecting CABC into goat intervertebral discs results in mild disc degeneration after 12 weeks. Spontaneous recovery or leveling off of the degeneration has been reported before and is relevant when the goat model is used in regeneration studies. Reproducibility of the induced degeneration is relevant as well. Methods. Twelve goats were used in this study. The development of degeneration was studied after the injection of 0.25 U/mL CABC intradiscally. The development of degenerative signs was studied after 18 (n = 6) and 26 (n = 6) weeks by means of radiograph, magnetic resonance imaging, macroscopic analysis, and histology and biochemical evaluation. The induced degeneration was compared with the results from a previous study, in which degeneration was induced similarly and analysis was performed after 12 weeks. Results. The severity of the degenerative signs was mild and was consequently present in all parameters analyzed. When compared with the results after 12 weeks, the degeneration was similar in the present study. Spontaneous recovery was not observed up to 26 weeks. Conclusion. The injection with CABC in the intervertebral disc reproducibly results in mild disc degeneration in the goat. These findings corroborate the goat model as a suitable large animal model to evaluate mild disc degeneration and potential new therapies.

WEE1 inhibition sensitizes osteosarcoma to radiotherapy

BackgroundThe use of radiotherapy in osteosarcoma (OS) is controversial due to its radioresistance. OS patients currently treated with radiotherapy generally are inoperable, have painful skeletal metastases, refuse surgery or have undergone an intralesional resection of the primary tumor. After irradiation-induced DNA damage, OS cells sustain a prolonged G2 cell cycle checkpoint arrest allowing DNA repair and evasion of cell death. Inhibition of WEE1 kinase leads to abrogation of the G2 arrest and could sensitize OS cells to irradiation induced cell death.MethodsWEE1 expression in OS was investigated by gene-expression data analysis and immunohistochemistry of tumor samples. WEE1 expression in OS cell lines and human osteoblasts was investigated by Western blot. The effect of WEE1 inhibition on the radiosensitivity of OS cells was assessed by cell viability and caspase activation analyses after combination treatment. The presence of DNA damage was visualized using immunofluorescence microscopy. Cell cycle effects were investigated by flow cytometry and WEE1 kinase regulation was analyzed by Western blot.ResultsWEE1 expression is found in the majority of tested OS tissue samples. Small molecule drug PD0166285 inhibits WEE1 kinase activity. In the presence of WEE1-inhibitor, irradiated cells fail to repair their damaged DNA, and show higher levels of caspase activation. The inhibition of WEE1 effectively abrogates the irradiation-induced G2 arrest in OS cells, forcing the cells into premature, catastrophic mitosis, thus enhancing cell death after irradiation treatment.ConclusionWe show that PD0166285, a small molecule WEE1 kinase inhibitor, can abrogate the G2 checkpoint in OS cells, pushing them into mitotic catastrophe and thus sensitizing OS cells to irradiation-induced cell death. This suggests that WEE1 inhibition may be a promising strategy to enhance the radiotherapy effect in patients with OS.

Human platelet lysate as a fetal bovine serum substitute improves human adipose-derived stromal cell culture for future cardiac repair applications

Adipose-derived stromal cells (ASC) are promising candidates for cell therapy, for example to treat myocardial infarction. Commonly, fetal bovine serum (FBS) is used in ASC culturing. However, FBS has several disadvantages. Its effects differ between batches and, when applied clinically, transmission of pathogens and antibody development against FBS are possible. In this study, we investigated whether FBS can be substituted by human platelet lysate (PL) in ASC culture, without affecting functional capacities particularly important for cardiac repair application of ASC. We found that PL-cultured ASC had a significant 3-fold increased proliferation rate and a significantly higher attachment to tissue culture plastic as well as to endothelial cells compared with FBS-cultured ASC. PL-cultured ASC remained a significant 25% smaller than FBS-cultured ASC. Both showed a comparable surface marker profile, with the exception of significantly higher levels of CD73, CD90, and CD166 on PL-cultured ASC. PL-cultured ASC showed a significantly higher migration rate compared with FBS-cultured ASC in a transwell assay. Finally, FBS- and PL-cultured ASC had a similar high capacity to differentiate towards cardiomyocytes. In conclusion, this study showed that culturing ASC is more favorable in PL-supplemented medium compared with FBS-supplemented medium.