In the United States, 1.5 million individuals suffer a fracture due to bone disease each year. In addition, there are many unknown mechanisms behind how muscular disorders and mechanical load adversely affect bone development, such as in the disease distal arthrogryposis. Disease research in human cell models has greater translational potential compared to animal models but have faced challenges when constructing highly-specialized tissues such as bone. We propose a novel, three-dimensional bone tissue model as a platform for musculoskeletal disease modeling that allows for compressive loading. By seeding induced pluripotent stem cell (iPSC) derived osteoblasts and osteoclasts in a 3D, porous, hydroxyapatite-coated poly-L-lactide scaffold, we propose to generate a bone tissue model that replicates human tissue in a laboratory. By applying compression to the novel 3D bone tissue model, we expect to observe phenotypes of bone disorders and bone development under mechanical loading. We propose to induce osteoblast and osteoclasts lineage from mesenchymal progenitor cells and hematopoietic progenitor cells, respectively, and co-culture to identify optimal conditions for cell growth. Preliminary experiments have found success in culturing active osteoblasts from iPSC-derived mesenchymal progenitor cells. By screening for markers of cell proliferation, calcium deposition, bone resorption and secretion, the cultures can be assessed for their robustness. In parallel, a porous scaffold will be fabricated by dissolving poly-L-lactide in chloroform and molding over sodium chloride particles. Coating said scaffold in fibronectin and hydroxyapatite will improve cell adhesion and uptake bone secretion. Seeding osteoclast and osteoblasts cells in a porous scaffold will allow for improved cell diffusion and 3D growth, mimicking the human microenvironment. We expect that combining robust, osteogenic tissue culture on a bioactive scaffold that allows 3D bone growth with mechanical loading will reveal phenotypes of distal arthrogryposis. Thus, this method has significant applications in accelerating laboratory findings to clinical research.

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) that have been engineered into three-dimensional heart tissues (EHTs) are valuable research tools for investigating debilitating genetic diseases that afflict the heart, such as Duchenne muscular dystrophy (DMD). Ensuring iPSC-CMs can be sufficiently matured to model such diseases remains a hurdle in current research, and maturational analysis techniques for iPSC-CMs are either qualitative, manual, or primarily based in two dimensions, leaving much to be desired. In this project, we created a suite of MATLAB image-processing scripts that can quantify the effect of three-dimensional culture and disease-causing DMD mutations on cardiomyocyte structure and maturation state. The iPSC-CMs were differentiated from stem cells, cast into EHTs, stained using immunofluorescence, and imaged using a confocal microscope. Using the scripts to analyze these 3D images of iPSC-CM stains, key maturational features of the cells can be quantified such as nuclei count; cardiomyocyte area; and sarcomere length, orientation, and z-disk width. Analyzing cardiomyocyte area can give key information on cardiomyocyte hypertrophy while examining sarcomere length, orientation, and Z-disk width can provide information on myofibril structure and organization. The suite allows analysis of these maturational features in both 2D and 3D cultures and offers a method for quantitatively assessing maturation in an automated manner. Measuring iPSC-CM maturation will also allow better comparison of existing maturational methods, such as mechanical loading, electrical stimulation, and small molecule treatment. The suite can also create graphical outputs to elegantly display data. Overall, the suite will help improve maturational analysis of EHTs, and hopefully contribute to the discovery of new treatments for diseases that affect the heart.