Kitsie Penick

FGF‐2 enhances the mitotic and chondrogenic potentials of human adult bone marrow‐derived mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) expanded with and without fibroblast growth factor (FGF) supplementation were compared with respect to their proliferation rate, ability to differentiate along the chondrogenic pathway in vitro, and their gene expression profiles. hMSCs expanded in FGF‐supplemented medium were smaller and proliferated more rapidly than hMSCs expanded in control conditions. Chondrogenic cultures made with FGF‐treated cells were larger and contain more proteoglycan than those made with control cells. Furthermore, aggregates of FGF‐treated cells lacked the collagen type I‐positive and collagen type II‐negative outer layer characteristic of aggregates of control cells. A total of 358 unique transcripts were differentially expressed in FGF‐treated hMSCs. Of these, 150 were upregulated and 208 downregulated. Seventeen percent of these genes affect proliferation. Known genes associated with cellular signaling functions comprised the largest percentage (∼20%) of differentially expressed transcripts. Eighty percent of differentially expressed extracellular matrix‐related genes were downregulated. The present findings that FGF‐2 enhances proliferation and differentiation of hMSCs adds to a growing body of evidence that cytokines modulate the differentiation potential and, perhaps, the multipotentiality of adult stem cells. With the generation of gene expression profiles of FGF‐treated and control cells we have taken the first steps in the elucidation of the molecular mechanism(s) behind these phenomena.

High-throughput aggregate culture system to assess the chondrogenic potential of mesenchymal stem cells

We have developed an improved method for preparing cell aggregates for in vitro chondrogenesis studies. This method is a modification of a previously developed conical tube-based culture system that replaces the original 15-mL polypropylene tubes with 96-well plates. These modifications allow a high-throughput approach to chondrogenic cultures, which reduces both the cost and time to produce chondrogenic aggregates, with no detrimental effects on the histological and histochemical qualities of the aggregates. We prepared aggregates in both systems with human bone marrow-derived mesenchymal stem cells (hMSC). The aggregates were harvested after 2 and 3 weeks in chondrogenic culture and analyzed for their ability to differentiate along the chondrogenic pathway in a defined in vitro environment. Chondrogenic differentiation was assessed biochemically by DNA and glycosaminoglycan (GAG) quantification assays and by histological and immunohistologic assessment. The chondrogenic cultures produced in the 96-well plates appear to be slightly larger in size and contain more DNA and GAG than the aggregates made in tubes. When analyzed histologically, both systems demonstrate morphological characteristics that are consistent with chondrogenic differentiation and cartilaginous extracellular matrix production.

Chondrogenic Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells: Tips and Tricks

It is well known that adult cartilage lacks the ability to repair itself; this makes articular cartilage a very attractive target for tissue engineering. The majority of articular cartilage repair models attempt to deliver or recruit reparative cells to the site of injury. A number of efforts are directed to the characterization of progenitor cells and the understanding of the mechanisms involved in their chondrogenic differentiation. Our laboratory has focused on cartilage repair using mesenchymal stem cells and studied their differentiation into cartilage. Mesenchymal stem cells are attractive candidates for cartilage repair due to their osteogenic and chondrogenic potential, ease of harvest, and ease of expansion in culture. However, the need for chondrogenic differentiation is superposed on other technical issues associated with cartilage repair; this adds a level of complexity over using mature chondrocytes. This chapter will focus on the methods involved in the isolation and expansion of human mesenchymal stem cells, their differentiation along the chondrogenic lineage, and the qualitative and quantitative assessment of chondrogenic differentiation.

Simplification of aggregate culture of human mesenchymal stem cells as a chondrogenic screening assay

Aggregate culture provides a three-dimensional (3-D) environment for differentiating or differentiated cells; it is particularly useful to study in vitro chondrogenesis and cartilage biology. We have recently ported this method from a conical tube-based format to a 96-well plate format for the study of mesenchymal stem cell (MSC) chondrogenesis. The microplate format has greatly reduced the workload and materials cost, while maintaining reproducible chondrogenic differentiation. A long-term goal is to fully automate aggregate culture--this requires critically identifying all the indispensable steps of the protocol. Robotic laboratory equipment for manipulating microplate assays are commercially available; however centrifugation steps are difficult to implement automatically. We, therefore, tested whether the centrifugation step can be eliminated, thus significantly streamlining the assay workflow. By comparing aggregates prepared from human bone marrow-derived MSCs (hMSCs) that were formed either through centrifugation or through free sedimentation, we found that both methods produce aggregates with similar formation kinetics, and that there was no perceptible difference in the timing of the appearance of markers of chondrogenesis. Thus, it appears safe to eliminate the centrifugation step from the aggregate culture protocol. This results in significant time and effort savings and paves the way for future full automation of the aggregate assay.

Concentrated collagen-chondroitin sulfate scaffolds for tissue engineering applications

Collagen-chondroitin sulfate biomaterial scaffolds have been used in a number of tissue-engineered products under development or in the clinics. In this article, we describe a new approach based on centrifugation for obtaining highly concentrated yet porous collagen scaffolds. Water uptake, chondroitin sulfate retention, morphology, mechanical properties, and tissue-engineering potential of the concentrated scaffolds were investigated. Our results show that the new approach can lead to scaffolds containing four times as much collagen as that in conventional unconcentrated scaffolds. Further, water uptake in the concentrated scaffolds was significantly greater while chondroitin sulfate retention in the concentrated scaffolds was unaffected. The value of mean pore diameter in the concentrated scaffolds was smaller than that in the unconcentrated scaffolds and the walls of the pores in the former comprised of a continuous sheet of collagen. The mechanical properties measured as moduli of elasticity in compression and tension were improved by as much as 30 times in the concentrated scaffolds. In addition, our tissue culture results with human mesenchymal stem cells and foreskin keratinocytes show that the new scaffolds can be used for cartilage and skin tissue-engineering applications.

Assessing adipogenic potential of mesenchymal stem cells: a rapid three-dimensional culture screening technique.

Bone-marrow-derived mesenchymal stem cells (MSCs) have the potential to differentiate into a number of phenotypes, including adipocytes. Adipogenic differentiation has traditionally been performed in monolayer culture, and, while the expression of a fat-cell phenotype can be achieved, this culture method is labor and material intensive and results in only small numbers of fragile adherent cells, which are not very useful for further applications. Aggregate culture is a cell-culture technique in which cells are induced to form three-dimensional aggregates; this method has previously been used successfully, among others, to induce and study chondrogenic differentiation of MSCs. We have previously published an adaptation of the chondrogenic aggregate culture method to a 96-well plate format. Based on the success of this method, we have used the same format for the preparation of three-dimensional adipogenic cultures. The MSCs differentiate rapidly, the aggregates can be handled and processed for histologic and biochemical assays with ease, and the format offers significant savings in supplies and labor. As a differentiation assay, this method can distinguish between degrees of senescence and appears suitable for testing medium or drug formulations in a high-volume, high-throughput fashion.

Rapid porcine lung decellularization using a novel organ regenerative control acquisition bioreactor

To regenerate discarded lungs that would not normally be used for transplant, ex vivo reseeding after decellularization may produce organs suitable for clinical transplantation and therefore close the donor gap. Organ regenerative control acquisition (Harvard Biosciences, Holliston, MA), a novel bioreactor system that simulates physiological conditions, was used to evaluate a method of rapid decellularization. Although most current decellularization methods are 24–72 hours, we hypothesized that perfusing porcine lungs with detergents at higher pressures for less time would yield comparable bioscaffolds suitable for future experimentation. Methods involved perfusion of 1% Triton X-100 (Triton) and 0.1% sodium dodecyl sulfate at varied physiological flow rates. Architecture of native and decellularized lungs was analyzed with hematoxylin and eosin (H&E) staining, transmission electron microscopy (TEM), and scanning electron microscopy (SEM). Dry gas and liquid ventilation techniques were introduced. Our 7 hour decellularization procedure removes nuclear material while maintaining architecture. Bioscaffolds have the microarchitecture for reseeding of stem cells. Hematoxylin and eosin staining suggested removal of nuclear material, whereas SEM and TEM imaging demonstrated total removal of cells with structural architecture preserved. This process can lead to clinical implementation, thereby increasing the availability of human lungs for transplantation.