The derivation of astrocytes from human pluripotent stem cells is currently slow and inefficient. We demonstrate that overexpression of the transcription factors SOX9 and NFIB in human pluripotent stem cells rapidly and efficiently yields homogeneous populations of induced astrocytes. In our study these cells exhibited molecular and functional properties resembling those of adult human astrocytes and were deemed suitable for disease modeling. Our method provides new possibilities for the study of human astrocytes in health and disease.

Several studies have reported endothelial cell (EC) derivation from human induced pluripotent stem cells (hiPSCs). However, few have explored their functional properties in depth with respect to line-to-line and batch-to-batch variability and how they relate to primary ECs. We therefore carried out accurate characterization of hiPSC-derived ECs (hiPSC-ECs) from multiple (non-integrating) hiPSC lines and compared them with primary ECs in various functional assays, which included barrier function using real-time impedance spectroscopy with an integrated assay of electric wound healing, endothelia-leukocyte interaction under physiological flow to mimic inflammation and angiogenic responses in in vitro and in vivo assays. Overall, we found many similarities but also some important differences between hiPSC-derived and primary ECs. Assessment of vasculogenic responses in vivo showed little difference between primary ECs and hiPSC-ECs with regard to functional blood vessel formation, which may be important in future regenerative medicine applications requiring vascularization.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) grown in engineered heart tissue (EHT) can be used for drug screening, disease modeling, and heart repair. However, the immaturity of hiPSC-CMs currently limits their use. Because mechanical loading increases during development and facilitates cardiac maturation, we hypothesized that afterload would promote maturation of EHTs. To test this we developed a system in which EHTs are suspended between a rigid post and a flexible one, whose resistance to contraction can be modulated by applying braces of varying length. These braces allow us to adjust afterload conditions over two orders of magnitude by increasing the flexible post resistance from 0.09 up to 9.2 mu$N/mu$m. After three weeks in culture, optical tracking of post deflections revealed that auxotonic twitch forces increased in correlation with the degree of afterload, whereas twitch velocities decreased with afterload. Consequently, the power and work of the EHTs were maximal under intermediate afterloads. When studied isometrically, the inotropy of EHTs increased with afterload up to an intermediate resistance (0.45 mu$N/mu$m) and then plateaued. Applied afterload increased sarcomere length, cardiomyocyte area and elongation, which are hallmarks of maturation. Furthermore, progressively increasing the level of afterload led to improved calcium handling, increased expression of several key markers of cardiac maturation, including a shift from fetal to adult ventricular myosin heavy chain isoforms. However, at the highest afterload condition, markers of pathological hypertrophy and fibrosis were also upregulated, although the bulk tissue stiffness remained the same for all levels of applied afterload tested. Together, our results indicate that application of moderate afterloads can substantially improve the maturation of hiPSC-CMs in EHTs, while high afterload conditions may mimic certain aspects of human cardiac pathology resulting from elevated mechanical overload.

Calcium/calmodulin-dependent protein kinase II (CAMK2) plays fundamental roles in synaptic plasticity that underlies learning and memory. Here, we describe a new recessive neurodevelopmental syndrome with global developmental delay, seizures and intellectual disability. Using linkage analysis and exome sequencing, we found that this disease maps to chromosome 5q31.1-q34 and is caused by a biallelic germline mutation in CAMK2A. The missense mutation, p.His477Tyr is located in the CAMK2A association domain that is critical for its function and localization. Biochemically, the p.His477Tyr mutant is defective in self-oligomerization and unable to assemble into the multimeric holoenzyme.In vivo, CAMK2AH477Y failed to rescue neuronal defects in C. elegans lacking unc-43, the ortholog of human CAMK2A. In vitro, neurons derived from patient iPSCs displayed profound synaptic defects. Together, our data demonstrate that a recessive germline mutation in CAMK2A leads to neurodevelopmental defects in humans and suggest that dysfunctional CAMK2 paralogs may contribute to other neurological disorders.

Mesenchymal stem cells (MSCs) have been isolated from various mesodermal and ectodermal tissues. While the phenotypic and functional heterogeneity of MSCs stemming from their developmental origins has been acknowledged, the genetic and environmental factors underpinning these differences are not well-understood. Here, we investigated whether substrate stiffness mediated mechanical cues can directly modulate the development of ectodermal MSCs (eMSCs) from a precursor human neural crest stem cell (NCSC) population. We showed that NCSC-derived eMSCs were transcriptionally and functionally distinct from mesodermal bone marrow MSCs. eMSCs derived on lower substrate stiffness specifically increased their expression of the MSC marker, CD44 in a Rho-ROCK signaling dependent manner, which resulted in a concomitant increase in the eMSCs' adipogenic and chondrogenic differentiation potential. This mechanically-induced effect can only be maintained for short-term upon switching back to a stiff substrate but can be sustained for longer-term when the eMSCs were exclusively maintained on soft substrates. We also discovered that CD44 expression modulated eMSC self-renewal and multipotency via the downregulation of downstream platelet-derived growth factor receptor beta (PDGFRbeta$) signaling. This is the first instance demonstrating that substrate stiffness not only influences the differentiation trajectories of MSCs but also their derivation from upstream progenitors, such as NCSCs.