Rigid microenvironments promote cardiac differentiation of mouse and human embryonic stem cells.

While adult heart muscle is the least regenerative of tissues, embryonic cardiomyocytes are proliferative, with embryonic stem (ES) cells providing an endless reservoir. In addition to secreted factors and cell-cell interactions, the extracellular microenvironment has been shown to play an important role in stem cell lineage specification, and understanding how scaffold elasticity influences cardiac differentiation is crucial to cardiac tissue engineering. Though previous studies have analyzed the role of the matrix elasticity on the function of differentiated cardiomyocytes, whether it affects the induction of cardiomyocytes from pluripotent stem cells is poorly understood. Here, we examined the role of matrix rigidity on the cardiac differentiation using mouse and human ES cells. Culture on polydimethylsiloxane (PDMS) substrates of varied monomer-to-crosslinker ratios revealed that rigid extracellular matrices promote a higher yield of de novo cardiomyocytes from undifferentiated ES cells. Using an genetically modified ES system that allows us to purify differentiated cardiomyocytes by drug selection, we demonstrate that rigid environments induce higher cardiac troponin T expression, beating rate of foci, and expression ratio of adult α- to fetal β- myosin heavy chain in a purified cardiac population. M-mode and mechanical interferometry image analyses demonstrate that these ES-derived cardiomyocytes display functional maturity and synchronization of beating when co-cultured with neonatal cardiomyocytes harvested from a developing embryo. Together, these data identify matrix stiffness as an independent factor that instructs not only the maturation of the already differentiated cardiomyocytes but also the induction and proliferation of cardiomyocytes from undifferentiated progenitors. Manipulation of the stiffness will help direct the production of functional cardiomyocytes en masse from stem cells for regenerative medicine purposes

Haemogenic endocardium contributes to transient definitive haematopoiesis.

Haematopoietic cells arise from spatiotemporally restricted domains in the developing embryo. Although studies of non-mammalian animal and in vitro embryonic stem cell models suggest a close relationship among cardiac, endocardial and haematopoietic lineages, it remains unknown whether the mammalian heart tube serves as a haemogenic organ akin to the dorsal aorta. Here we examine the haemogenic activity of the developing endocardium. Mouse heart explants generate myeloid and erythroid colonies in the absence of circulation. Haemogenic activity arises from a subset of endocardial cells in the outflow cushion and atria earlier than in the aorta-gonad-mesonephros region, and is transient and definitive in nature. Interestingly, key cardiac transcription factors, Nkx2-5 and Isl1, are expressed in and required for the haemogenic population of the endocardium. Together, these data suggest that a subset of endocardial/endothelial cells serve as a de novo source for transient definitive haematopoietic progenitors

Combined Particulated Juvenile Cartilage Allograft Transplantation and Medial Patellofemoral Ligament Reconstruction for Symptomatic Chondral Defects in the Setting of Recurrent Patellar Instability

Successful management of patellar osteochondral lesions has proved difficult, with unreliable outcomes reported using traditional cartilage repair and restoration procedures. Unique considerations for this type of defect include the multiplanar contours of the articular surface of the patella, high compressive and shear forces with knee range of motion that may disrupt graft healing, and the potential need for concomitant surgery to address patellar malalignment and instability. We describe our preferred method for treatment of a symptomatic chondral defect in the setting of recurrent patellar instability using particulated juvenile articular cartilage allograft transplantation and medial patellofemoral ligament reconstruction with semitendinosus allograft. Distinct advantages of this cartilage restoration technique include single-stage restoration of relevant cartilage pathology and the ability to easily contour the graft to the size and shape of the chondral defect