Developing an optimized growth environment for adipose-derived stems cells (ASCs) to obtain clinically useable cell quantities from relatively small tissue biopsies would significantly impact the field of tissue engineering. To date, ASCs have been differentiated into adipose, bone, cartilage, smooth muscle, endothelial, skeletal muscle, nervous, and cardiac tissue. Therefore, ASCs have potential for use in the treatment of a wide variety of clinical conditions ranging from myocardial infarction, to musculoskeletal disorders, and the repair of soft tissue defects. In this work, a custom-designed, 3-dimensional (3-D) scaffold-based perfusion bioreactor system was investigated in the culture of ASCs. Decellularized adipose tissue (DAT) was used to provide a 3-dimensional scaffold, as it possesses the native extracellular matrix (ECM) architecture and composition of human adipose tissue. The DAT had a permeability of 149 m2, based on a perfusion rate of 1.5 mL/min over a 200 mg DAT sample, and the culturing medium was evenly perfused throughout the DAT, thereby permitting possible cell growth within the central regions. Initial culturing studies of human ASCs on tissue culture polystyrene (TCPS) demonstrated that hypoxic (5% O2) conditions decreased the doubling time, and resulted in enhanced cell proliferation, as compared to normoxic (21% O2) conditions. The cell imaging and DNA quantification results showed that suspension seeding of the ASCs permitted cell attachment to the DAT scaffold, but did not support long-term ASC growth. In contrast, when the ASCs were seeded as multicellular aggregates, the cells attached and underwent measurable proliferation. The optimal seeding density observed was 1 x 106 ASCs/scaffold; or 50 aggregates (20,000 ASCs/aggregate) per scaffold. Based on the confocal imaging, the ASCs remained spherical in morphology during the entire culturing period. Moreover, results illustrated that the perfusion bioreactor provided an improved culturing environment for ASCs over traditional static culturing. Hypoxic (5% O2) conditions showed improved proliferation over normoxic (21% O2) conditions, within the bioreactor system. After a 14-day hypoxic culturing period in the perfusion bioreactor, the seeded ASCs retained the ability to undergo adipogenesis, as indicated by Glycerol-3-Phsophate Dehydrogenase (GPDH) enzymatic activity measurements, demonstrating the promise of this approach for soft tissue engineering applications / Thesis (Master, Chemical Engineering) -- Queen's University, 2011-11-09 20:28:34.252

Despite the importance of inter-cellular (between cell) communication networks in regulating homeostasis in multicellular organisms, very little is known about their topology, dynamics, or functional significance. Inter-cellular communication networks are particularly relevant in stem cell biology, as stem cell fate decisions (self-renewal, proliferation, lineage specification) are tightly regulated based on physiological demand. Using human blood stem cell cultures as an experimental paradigm, we present an integrated experimental and computational approach to interrogate a hierarchically organized tissue network. We have developed a novel mathematical model of blood stem cell development incorporating cell-level kinetic parameters as functions of secreted molecule-mediated inter-cellular networks. By relation to quantitative cellular assays, our model is capable of predictively simulating many disparate features of both normal and malignant hematopoiesis, relating internal parameters and microenvironmental variables to measurable cell fate outcomes. Through integrated in silico and experimental analyses we show blood stem and progenitor cell fate is regulated by cell-cell feedback, and can be controlled non-cell autonomously by dynamically perturbing inter-cellular signalling. Furthermore, we have compiled genome-scale molecular profiles (transcriptome and secretome), publicly available databases, and literature mining to reconstruct soluble factor-mediated inter-cellular signalling networks regulating cell fate decisions. We find that dynamic interactions between positive and negative regulators, in the context of tuneable cell culture parameters, tip the balance between stem cell supportive vs. non-supportive conditions. The cell-cytokine interactions can be summarized as an antagonistic positive-negative feedback circuit wherein stem cell self-renewal is regulated by a balance of megakaryocyte-derived stimulatory factors vs. monocyte-derived inhibitory factors. To understand how the experimentally identified positive and negative regulatory signals are integrated at the intra-cellular level, we define a literature-derived blood stem cell self-renewal network wherein these extracellular signals converge for coherent processing into cell fate decisions. In summary, this work demonstrates the utility of integrating experimental and computational methods to explore complex cellular systems, and represents the first attempt to comprehensively elucidate non-autonomous signals balancing stem cell homeostasis and regeneration.

Human embryonic stem cells (hESCs) replicate in vitro by the process of self-renewal, whilst maintaining their pluripotency. Understanding the pathways involved in the regulation of this process will assist in developing fully-defined conditions for the robust proliferation of hESCs necessary for therapeutic applications.

No description available.

In this work, I investigated the influence of different surfaces on stem cell proliferation and osteogenetic differentiation. Surface properties of biomaterials are important factors that influence cell fate such as cell adhesion, viability, proliferation and differentiation. Herein, mesenchymal stem cells (MSCs) were cultured on composite electrospun nanofibrous membranes with varied surface wettability for designed periods and cell morphologies, proliferation and viability were characterized via analysis methods such as Infrared attenuated total reflectance Spectroscopy (IR-ATR), scanning electron microscopy (SEM) and MTT cell proliferation assay. The expression of genes associated with osteogenesis, including bone sialoprotein (BSP), alkaline phosphatase (ALP), osteopontin (OPN) and osteocalcin (OCN) were measured by real-time RT-PCR on different time points. Through western blot analysis, ERK1/2 pathway was found to be responsible for the differentiation of MSCs on nanofibrous membranes with different wettability.

We have studied the factors that are important for producing functional retinal neurons from adult derived retinal stem and progenitor cells (RPCs) investigating the role of substrate stiffness or soluble factors in 2D and 3D culture on the differentiation and survival of RPCs. RPCs were cultured on agarose scaffolds modified with the adhesive peptides of varying stiffness. We observed that cell survival in the 0.75% matrix was greater in 3D than in 2D by a factor of 50% irrespective of the time in culture. We observed the presence of photoreceptors exclusively in 0.75% agarose, while the stiffer matrices (1.75%) led to retinal ganglion cell and glial cell differentiation with no significant difference in the differentiation profiles when cells were cultured in 2D vs. 3D. These data indicate that substrate stiffness, more than growth factors, has a significant impact on both the survival and differentiation profile of retinal precursor cells.

We have studied the factors that are important for producing functional retinal neurons from adult derived retinal stem and progenitor cells (RPCs) investigating the role of substrate stiffness or soluble factors in 2D and 3D culture on the differentiation and survival of RPCs. RPCs were cultured on agarose scaffolds modified with the adhesive peptides of varying stiffness. We observed that cell survival in the 0.75% matrix was greater in 3D than in 2D by a factor of 50% irrespective of the time in culture. We observed the presence of photoreceptors exclusively in 0.75% agarose, while the stiffer matrices (1.75%) led to retinal ganglion cell and glial cell differentiation with no significant difference in the differentiation profiles when cells were cultured in 2D vs. 3D. These data indicate that substrate stiffness, more than growth factors, has a significant impact on both the survival and differentiation profile of retinal precursor cells.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2001. / Härtill 6 uppsatser.

No description available.