Cellular viability was demonstrated in both TheraSkin and fresh skin.

While induced pluripotent stem cells (iPSCs) hold great clinical promise, one hurdle that remains is the existence of a parental germ-layer memory in reprogrammed cells leading to preferential differentiation fates. While it is problematic for generating cells vastly different from the reprogrammed cells' origins, it could be advantageous for the reliable generation of germ-layer specific cell types for future therapeutic use. Here we use human osteoblast-derived iPSCs (hOB-iPSCs) to generate induced osteoprogenitors (iOPs). Osteoblasts were successfully reprogrammed and demonstrated by endogenous upregulation of Oct4, Sox2, Nanog, TRA-1-81, TRA-16-1, SSEA3, and confirmatory hPSC Scorecard Algorithmic Assessment. The hOB-iPSCs formed embryoid bodies with cells of ectoderm and mesoderm but have low capacity to form endodermal cells. Differentiation into osteoprogenitors occurred within only 2–6 days, with a population doubling rate of less than 24 hrs; however, hOB-iPSC derived osteoprogenitors were only able to form osteogenic and chondrogenic cells but not adipogenic cells. Consistent with this, hOB-iOPs were found to have higher methylation of PPARγ but similar levels of methylation on the RUNX2 promoter. These data demonstrate that iPSCs can be generated from human osteoblasts, but variant methylation patterns affect their differentiation capacities. Therefore, epigenetic memory can be exploited for efficient generation of clinically relevant quantities of osteoprogenitor cells.

DBM fibers can be engineered into custom-shaped, osteoinductive and osteoconductive implants with potential for repairing osseous defects with precise fitment, potentially reducing operating time. By providing pre-formed and custom implants, this regenerative allograft may improve patient outcomes following surgical bone repair, while further advancing personalized orthopedic and craniomaxillofacial medicine using three-dimensional-printed tissue molds.

Three-dimensional bioprinted culture platforms mimic the native microenvironment of tissues more accurately than two-dimensional cell cultures or animal models. Scaffold-free bioprinting eliminates many complications associated with traditional scaffold-dependent printing as well as provides better cell-to-cell interactions and long-term functionality. In this study, constructs were produced from bone marrow derived mesenchymal stem cells (BM-MSCs) using a scaffold-free bioprinter. These constructs were cultured in either osteogenic, chondrogenic, a 50:50 mixture of osteogenic and chondrogenic (‘osteo-chondro’), or BM-MSC growth medium. Osteogenic and chondrogenic differentiation capacity was determined over an 8-week culture period using histological and immunohistochemical staining and RT-qPCR (Phase I). After 6 weeks in culture, individual osteogenic and chondrogenic differentiated constructs were adhered to create a bone-cartilage interaction model. Adhered differentiated constructs were cultured for an additional 8 weeks in either chondrogenic or osteo-chondro medium to evaluate sustainability of lineage specification and transdifferentiation potential (Phase II). Constructs cultured in their respective osteogenic and/or chondrogenic medium differentiated directly into bone (model of intramembranous ossification) or cartilage. Positive histological and immunohistochemical staining for bone or cartilage identification was shown after 4 and 8 weeks in culture. Expression of osteogenesis and chondrogenesis associated genes increased between weeks 2 and 6. Adhered individual osteogenic and chondrogenic differentiated constructs sustained their differentiated phenotype when cultured in chondrogenic medium. However, adhered individual chondrogenic differentiated constructs cultured in osteo-chondro medium were converted to bone (model of metaplastic transformation). These bioprinted models of bone-cartilage interaction, intramembranous ossification, and metaplastic transformation of cartilage into bone offer a useful and promising approach for bone and cartilage tissue engineering research. Specifically, these models can be potentially used as functional tissue systems for studying osteochondral defect repair, drug discovery and response, and many other potential applications.

Human pluripotent stem cells (hPSCs) hold great promise for future applications in drug discovery and cell therapies. hPSC culture protocols require specific substrates and medium supplements to support cell expansion and lineage specific differentiation. The animal origin of these substrates is a severe limitation when considering the translation of hPSC derivatives to the clinic and in vitro disease modeling. The present study evaluates the use of a human placenta-derived extracellular matrix (ECM) hydrogel, HuGentra Ⓡ , to support tri-lineage differentiation of human induced pluripotent stem cells (hiPSCs). Lineage-specific embryoid bodies (EBs) were plated onto three separate matrices, and differentiation efficiency was evaluated based on morphology, protein, and gene expression. HuGentra was found to support the differentiation of hiPSCs to all three germ layers: ectodermal, mesodermal, and endodermal lineages. hiPSCs differentiated into neurons, cardiomyocytes, and hepatocytes on HuGentra had similar morphology, protein, and gene expression compared to differentiation on Matrigel or other cell preferred matrices. HuGentra can be considered as a suitable human substrate for hiPSC differentiation.