A simple method for deriving functional MSCs and applied for osteogenesis in 3D scaffolds

We describe a simple method for bone engineering using biodegradable scaffolds with mesenchymal stem cells derived from human induced-pluripotent stem cells (hiPS-MSCs). The hiPS-MSCs expressed mesenchymal markers (CD90, CD73, and CD105), possessed multipotency characterized by tri-lineages differentiation: osteogenic, adipogenic, and chondrogenic, and lost pluripotency – as seen with the loss of markers OCT3/4 and TRA-1-81 – and tumorigenicity. However, these iPS-MSCs are still positive for marker NANOG. We further explored the osteogenic potential of the hiPS-MSCs in synthetic polymer polycaprolactone (PCL) scaffolds or PCL scaffolds functionalized with natural polymer hyaluronan and ceramic TCP (PHT) both in vitro and in vivo. Our results showed that these iPS-MSCs are functionally compatible with the two 3D scaffolds tested and formed typically calcified structure in the scaffolds. Overall, our results suggest the iPS-MSCs derived by this simple method retain fully osteogenic function and provide a new solution towards personalized orthopedic therapy in the future

Protocol for generating reproducible miniaturized controlled midbrain organoids

Summary: Here, we present a protocol for generating miniaturized controlled midbrain organoids (MiCOs) of reproducible size and cellular composition, without a necrotic center. We describe steps for maintaining and passaging human pluripotent stem cells, generating MiCOs using AggreWellTM400, and maintaining them in an EB-Disk360on an orbital shaker, eliminating the need for Matrigel or a spinner flask and preventing organoid fusion. We then detail organoid collection for different endpoint analysis. This protocol is suitable for compound screening and disease modeling studies. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics