Human preadipocyte proliferation and differentiation

Obesity represents a major health, social and economic burden to many developing and Westernized communities, with the prevalence increasing at a rate exceeding almost all other medical conditions. Despite major recent advances in our understanding of adipose tissue metabolism and dynamics, we still have limited insight into the regulation of adipose tissue mass in humans. Any significant increase in adipose tissue mass requires proliferation and differentiation of precursor cells (preadipocytes) present in the stromo-vascular compartment of adipose tissue. These processes are very complex and an increasing number of growth factors and hormones have been shown to modulate the expression of genes involved in preadipocyte proliferation and differentiation. A number of transcription factors, including the C/EBP family and PP ARy, have been identified as integral to adipose tissue development and preadipocyte differentiation. Together PP ARy and C/EBPa regulate important events in the activation and maintenance of the terminally differentiated phenotype. The ability of PP ARy to increase transcription through its DNA recognition site is dependent on the binding of ligands. This suggests that an endogenous PP ARy ligand may be an important regulator of adipogenesis. Adipose tissue functions as both the major site of energy storage in the body and as an endocrine organ synthesizing and secreting a number of important molecules involved in regulation of energy balance. For optimum functioning therefore, adipose tissue requires extensive vascularization and previous studies have shown that growth of adipose tissue is preceded by development of a microvascular network. This suggests that paracrine interactions between constituent cells in adipose tissue may be involved in both new capillary formation and fat cell growth. To address this hypothesis the work in this project was aimed at (a) further development of a method for inducing preadipocyte differentiation in subcultured human cells; (b) establishing a method for simultaneous isolation and separate culture of both preadipocytes and microvascular endothelial cells from the same adipose tissue biopsies; (c) to determine, using conditioned medium and co-culture techniques, if endothelial cell-derived factors influence the proliferation and/or differentiation of human preadipocytes; and (d) commence characterization of factors that may be responsible for any observed paracrine effects on aspects of human adipogenesis. Major findings of these studies were as follows: (A) Inclusion of either linoleic acid (a long-chain fatty acid reported to be a naturally occurring ligand for PP ARy) or Rosiglitazone (a member of the thiazolidinedione class of insulin-sensitizing drugs and a synthetic PPARy ligand) in differentiation medium had markedly different effects on preadipocyte differentiation. These studies showed that human preadipocytes have the potential to accumulate triacylglycerol irrespective of their stage of biochemical differentiation, and that thiazolidinediones and fatty acids may exert their adipogenic and lipogenic effects via different biochemical pathways. It was concluded that Rosiglitazone is a more potent inducer of human preadipocyte differentiation than linoleic acid. (B) A method for isolation and culture of both endothelial cells and preadipocytes from the same adipose tissue biopsy was developed. Adipose-derived microvascular endothelial cells were found to produce factor/s, which enhance both proliferation and differentiation of human preadipocytes. (C) The adipogenic effects of microvascular endothelial cells can be mimicked by exposure of preadipocytes to members of the Fibroblast Growth Factor family, specifically ~-ECGF and FGF-1. (D) Co-culture of human preadipocytes with endothelial cells or exposure of preadipocytes to either ~-ECGF or FGF-1 were found to 'prime' human preadipocytes, during their proliferative phase of growth, for thiazolidinedione-induced differentiation. (E) FGF -1 was not found to be acting as a ligand for PP ARy in this system. Findings from this project represent a significant step forward in our understanding of factors involved in growth of human adipose tissue and may lead to the development of therapeutic strategies aimed at modifying the process. Such strategies would have potential clinical utility in the treatment of obesity and obesity related disorders such as Type II Diabetes.

Repair of calvarial defects with customized tissue-engineered bone grafts - I. Evaluation of osteogenesis in a three-dimensional culture system

Bone generation by autogenous cell transplantation in combination with a biodegradable scaffold is one of the most promising techniques being developed in craniofacial surgery. The objective of this combined in vitro and in vivo study was to evaluate the morphology and osteogenic differentiation of bone marrow derived mesenchymal progenitor cells and calvarial osteoblasts in a two-dimensional (2-D) and three-dimensional (3-D) culture environment (Part I of this study) and their potential in combination with a biodegradable scaffold to reconstruct critical-size calvarial defects in an autologous animal model [Part II of this study; see Schantz, J.T., et al. Tissue Eng. 2003;9(Suppl. 1):S-127-S-139; this issue]. New Zealand White rabbits were used to isolate osteoblasts from calvarial bone chips and bone marrow stromal cells from iliac crest bone marrow aspirates. Multilineage differentiation potential was evaluated in a 2-D culture setting. After amplification, the cells were seeded within a fibrin matrix into a 3-D polycaprolactone (PCL) scaffold system. The constructs were cultured for up to 3 weeks in vitro and assayed for cell attachment and proliferation using phase-contrast light, confocal laser, and scanning electron microscopy and the MTS cell metabolic assay. Osteogenic differentiation was analyzed by determining the expression of alkaline phosphatase (ALP) and osteocalcin. The bone marrow-derived progenitor cells demonstrated the potential to be induced to the osteogenic, adipogenic, and chondrogenic pathways. In a 3-D environment, cell-seeded PCL scaffolds evaluated by confocal laser microscopy revealed continuous cell proliferation and homogeneous cell distribution within the PCL scaffolds. On osteogenic induction mesenchymal progenitor cells (12 U/L) produce significantly higher (p < 0.05) ALP activity than do osteoblasts (2 U/L); however, no significant differences were found in osteocalcin expression. In conclusion, this study showed that the combination of a mechanically stable synthetic framework (PCL scaffolds) and a biomimetic hydrogel (fibrin glue) provides a potential matrix for bone tissue-engineering applications. Comparison of osteogenic differentiation between the two mesenchymal cell sources revealed a similar pattern.

Colonization and osteogenic differentiation of different stem cell sources on electrospun nanofiber meshes

Numerous challenges remain in the successful clinical translation of cell-based therapies for musculoskeletal tissue repair, including the identification of an appropriate cell source and a viable cell delivery system. The aim of this study was to investigate the attachment, colonization, and osteogenic differentiation of two stem cell types, human mesenchymal stem cells (hMSCs) and human amniotic fluid stem (hAFS) cells, on electrospun nanofiber meshes. We demonstrate that nanofiber meshes are able to support these cell functions robustly, with both cell types demonstrating strong osteogenic potential. Differences in the kinetics of osteogenic differentiation were observed between hMSCs and hAFS cells, with the hAFS cells displaying a delayed alkaline phosphatase peak, but elevated mineral deposition, compared to hMSCs. We also compared the cell behavior on nanofiber meshes to that on tissue culture plastic, and observed that there is delayed initial attachment and proliferation on meshes, but enhanced mineralization at a later time point. Finally, cell-seeded nanofiber meshes were found to be effective in colonizing three-dimensional scaffolds in an in vitro system. This study provides support for the use of the nanofiber mesh as a model surface for cell culture in vitro, and a cell delivery vehicle for the repair of bone defects in vivo.

Differentially Expressed Protein Profile of Human Dental Pulp Cells in the Early Process of Odontoblast-like Differentiation In Vitro

Dental pulp cells (DPCs) are capable of differentiating into odontoblasts that secrete reparative dentin after pulp injury. The molecular mechanisms governing reparative dentinogenesis are yet to be fully understood. Here we investigated the differential protein profile of human DPCs undergoing odontogenic induction for 7 days. Using two-dimensional differential gel electrophoresis coupled with matrix-assisted laser adsorption ionization time of flight mass spectrometry, 2 3 protein spots related to the early odontogenic differentiation were identified. These proteins included cytoskeleton proteins, nuclear proteins, cell membrane-bound molecules, proteins involved in matrix synthesis, and metabolic enzymes. The expression of four identified proteins, which were heteronuclear ribonuclear proteins C, annexin VI, collagen type VI, and matrilin-2, was confirmed by Western blot and real-time realtime polymerase chain reaction analyses. This study generated a proteome reference map during odontoblast- like differentiation of human DPCs, which will be valuable to better understand the underlying molecular mechanisms in odontoblast-like differentiation.

Differences between in vitro viability and differentiation and in vivo bone-forming efficacy of human mesenchymal stem cells cultured on PCL-TCP scaffolds

Human mesenchymal stem cells (hMSCs) possess great therapeutic potential for the treatment of bone disease and fracture non-union. Too often however, in vitro evidence alone of the interaction between hMSCs and the biomaterial of choice is used as justification for continued development of the material into the clinic. Clearly for hMSC-based regenerative medicine to be successful for the treatment of orthopaedic trauma, it is crucial to transplant hMSCs with a suitable carrier that facilitates their survival, optimal proliferation and osteogenic differentiation in vitro and in vivo. This motivated us to evaluate the use of polycaprolactone-20% tricalcium phosphate (PCL-TCP) scaffolds produced by fused deposition modeling for the delivery of hMSCs. When hMSCs were cultured on the PCL-TCP scaffolds and imaged by a combination of phase contrast, scanning electron and confocal laser microscopy, we observed five distinct stages of colonization over a 21-day period that were characterized by cell attachment, spreading, cellular bridging, the formation of a dense cellular mass and the accumulation of a mineralized extracellular matrix when induced with osteogenic stimulants. Having established that PCL-TCP scaffolds are able to support hMSC proliferation and osteogenic differentiation, we next tested the in vivo efficacy of hMSC-loaded PCL-TCP scaffolds in nude rat critical-sized femoral defects. We found that fluorescently labeled hMSCs survived in the defect site for up to 3 weeks post-transplantation. However, only 50% of the femoral defects treated with hMSCs responded favorably as determined by new bone volume. As such, we show that verification of hMSC viability and differentiation in vitro is not sufficient to predict the efficacy of transplanted stem cells to consistently promote bone formation in orthotopic defects in vivo.

Differentiation of Rice Tungro Bacilliform Virus Strains by Restriction Analysis and DNA Hybridization

Rice tungro bacilliform virus (RTBV) is one of the two viruses that cause tungro disease. Four RTBV strains maintained in the greenhouse for 4 years, G1, G2, Ic, and L, were differentiated by restriction fragment length polymorphism (RFLP) analysis of the native viral DNA. Although strains G1 and Ic had identical restriction patterns when cleaved with Pst1, BamHI, EcoRI, and EcoRV, they can be differentiated from strains G2 and L by EcoRI and EcoRV digestion. These same endonucleases also differentiate strain G2 from strain L. When total DNA extracts from infected plants were used instead of viral DNA, and digested with EcoRV, identical restriction patterns for each strain (G2 and L) were obtained from roots, leaves, and leaf sheaths of infected plants. The restriction patterns were consistent from plant to plant, in different varieties, and at different times after inoculation. This technique can be used to differentiate RTBV strains and determine the variability of a large number of field samples.

Differentiation of Rice Tungro Spherical Virus Variants by RTPCR and RFLP

Differentiation of rice tungro spherical virus variants by RTPCR and RFLP tungro bacilliform virus (RTBV), the other causal agent, which causes the symptoms. RTSV is a single-stranded RNA virus of 12,180 nucleotides (Hull 1996).

Cell sourcing for bone tissue engineering : amniotic fluid stem cells have a delayed, robust differentiation compared to mesenchymal stem cells

Cell based therapies for bone regeneration are an exciting emerging technology, but the availability of osteogenic cells is limited and an ideal cell source has not been identified. Amniotic fluid-derived stem (AFS) cells and bone-marrow derived mesenchymal stem cells (MSCs) were compared to determine their osteogenic differentiation capacity in both 2D and 3D environments. In 2D culture, the AFS cells produced more mineralized matrix but delayed peaks in osteogenic markers. Cells were also cultured on 3D scaffolds constructed of poly-e-caprolactone for 15 weeks. MSCs differentiated more quickly than AFS cells on 3D scaffolds, but mineralized matrix production slowed considerably after 5 weeks. In contrast, the rate of AFS cell mineralization continued to increase out to 15 weeks, at which time AFS constructs contained 5-fold more mineralized matrix than MSC constructs. Therefore, cell source should be taken into consideration when used for cell therapy, as the MSCs would be a good choice for immediate matrix production, but the AFS cells would continue robust mineralization for an extended period of time. This study demonstrates that stem cell source can dramatically influence the magnitude and rate of osteogenic differentiation in vitro.

Application of hyperbaric oxygen in bone tissue engineering : effect of hyperbaric oxygen treatment on bone marrow stem cells

and non-union of bony fractures has been proposed since 1966, little has been known about the effect of HBOT on bone marrow stem cells (BMSC). The aim of this study is to investigate the effect of HBO treatment on osteogenetic differentiation of BMSC and potential application in bone tissue engineering. Adhesive stromal cells harvested from bone marrow were characterized by mesenchymal differentiation potential, cell surface markers and their proliferation capacity. Mesenchymal stem cells, which demonstrated osteogenic, chondrogenic and adipogenic differentiation potential and expressed positively for CD 29, CD 44, CD 73, CD 90, CD 105, CD 166 and negatively for CD34 and CD 45, were selected and treated in a laboratory-scale HBO chamber using different oxygen pressures and exposure times. No obvious effect of HBO treatment on BMSC proliferation was noticed. However, cytotoxic effects of HBO were considerably less pronounced when cells were cultured in medium supplemented with 10% FBS in comparison to medium supplemented with 2% FCS, as was evaluated by WST-1 assay. Under HBO treatment, bone nodules were formed in three days, which was clearly revealed by Von Kossa staining. In contrasts, without HBO treatment, bone nodules were not detected until 9-12 days using the same inducing culture media. Calcium deposition was also significantly increased after three days of HBO treatments compared to no HBO treatment. In addition it was also found that oxygen played a direct role in the enhancement of BMSC osteogenic differentiation, which was independent of the effect of air pressure.