Effects of ß-TCP scaffolds on neurogenic and osteogenic differentiation of human embryonic stem cells.

Extracellular matrix (ECM) and adhesion molecules play crucial roles in regulating growth and differentiation of stem cells. The current study aimed to investigate the effects of beta-tricalcium phosphate (ß-TCP) scaffolds on differentiation and expression of ECM and adhesion molecules of human embryonic stem cells (hESCs). Undifferentiated hESCs were seeded on ß-TCP scaffolds and cell culture plates and cultured in growth and osteogenic medium for 21 days. Scanning electron microscopy (SEM) displayed adhesion and growth of hESCs on the porous ß-TCP scaffolds. Histological analysis, immunohistochemical staining and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) demonstrated that the scaffolds supported growth and differentiation of hESCs. Expression levels of neural crest related genes (AP2a, FoxD3, HNK1, P75, Sox1, Sox10) and osteoblast-related genes (Runx2, SPP1 and BGLA) on the scaffolds in osteogenic medium were significantly higher than on the scaffolds in growth and cell culture plates in osteogenic medium, respectively (p<0.05). Polymerase chain reaction array experiments demonstrated increased expression of ECM and adhesion molecule-related genes on the scaffolds. In conclusion, osteoconductive scaffolds such as ß-TCP scaffolds promoted differentiation of hESCs, particularly expression of genes related to neural crest stem cell and osteoblastic differentiations. Beta-TCP scaffolds could be an alternative cell culture substrate for neural crest and osteogenic differentiation of hESCs. Optimization of culture medium may be necessary to enhance lineage restriction of hESCs on the ß-TCP scaffolds.

Phenotypic and differentiation stability of human embryonic stem cell-derived osteoblasts.

PURPOSE: To ensure the efficiency and safety of transplanted human embryonic stem cell (hESC)-derived osteoblast-like cells (hESC-OS) for bone regeneration, this study was designed to determine the effects of continuous cell expansion on the osteoblastic differentiation stability, pluripotency, and tumorigenic potential of long-term expanded hESC-OS. METHODS: hESCs manually harvested as cell aggregates or enzymatically dissociated as single cells were directly incubated in osteogenic medium and serially passaged to passage 25. Expression of osteoblast-related genes, pluripotent regulator genes, and genes related to tumorigenesis were examined at the primary passage and every 5 passages thereafter. hESC-OS were subcutaneously transplanted into nude mice for 4-24 weeks to test for teratoma formation. hESC-OS were recultivated in hESC culture conditions to evaluate the extent to which reverse differentiation back to the undifferentiated stage may occur. RESULTS: hESC-OS derived from hESC aggregates and dissociated cells exhibited comparable osteoblast differentiation patterns. Expression levels of osteoblast-related genes reached plateau levels at passages 5-10 before declining in higher passages. Expression of tumor-associated genes was not significantly increased. Only hESC-OS at primary and first passages formed teratomas after 4 weeks in vivo. The hESC-OS were not able to revert to hESCs. CONCLUSIONS: Expanded hESC-OS demonstrated lineage-specific differentiation stability, did not maintain the pluripotency of hES cells, and were genetically stable. Thus, hESC-OS may be considered for large animal preclinical studies.

Expansion and characterization of human embryonic stem cell-derived osteoblast-like cells.

Human embryonic stem cells (hESCs) have the potential to serve as a repository of cells for the replacement of damaged or diseased tissues and organs. However, to use hESCs in clinically relevant scenarios, a large number of cells are likely to be required. The aim of this study was to demonstrate an alternative cell culture method to increase the quantity of osteoblast-like cells directly derived from hESCs (hESCs-OS). Undifferentiated hESCs were directly cultivated and serially passaged in osteogenic medium (hESC-OS), and exhibited similar expression patterns of osteoblast-related genes to osteoblast-like cells derived from mesenchymal stem cells derived from hESCs (hESCs-MSCs-OS) and human bone marrow stromal cells (hBMSCs-OS). In comparison to hESCs-MSCs-OS, the hESCs-OS required a shorter expansion time to generate a homogenous population of osteoblast-like cells that did not contain contaminating undifferentiated hESCs. Identification of human specific nuclear antigen (HuNu) in the newly formed bone in calvarial defects verified the role of the transplanted hESCs-OS as active bone forming cells in vivo. Taken together, this study suggests that osteoblast-like cells directly derived from hESCs have the potential to serve as an alternative source of osteoprogenitors for bone tissue engineering strategies.

Isolation and characterization of novel atrazine-degrading microorganisms from an agricultural soil.

Six previously undescribed microorganisms capable of atrazine degradation were isolated from an agricultural soil that received repeated exposures of the commonly used herbicides atrazine and acetochlor. These isolates are all Gram-positive and group with microorganisms in the genera Nocardioides and Arthrobacter, both of which contain previously described atrazine degraders. All six isolates were capable of utilizing atrazine as a sole nitrogen source when provided with glucose as a separate carbon source. Under the culture conditions used, none of the isolates could utilize atrazine as the sole carbon and nitrogen source. We used several polymerase-chain-reaction-based assays to screen for the presence of a number of atrazine-degrading genes and verified their identity through sequencing. All six isolates contain trzN and atzC, two well-characterized genes involved in the conversion of atrazine to cyanuric acid. An additional atrazine-degrading gene, atzB, was detected in one of the isolates as well, yet none appeared to contain atzA, a commonly encountered gene in atrazine impacted soils and atrazine-degrading isolates. Interestingly, the deoxyribonucleic acid sequences of trzN and atzC were all identical, implying that their presence may be the result of horizontal gene transfer among these isolates.