Integrative Analysis of Long Isoform Sequencing and Functional Data Identifies Distinct Cortical Layer Neuronal Subtypes Derived from Human iPSCs.

Generation of human induced pluripotent stem cells (iPSCs) through reprogramming was a transformational change in the field of regenerative medicine that led to new possibilities for drug discovery and cell replacement therapy. Several protocols have been established to differentiate hiPSCs into neuronal lineages. However, low differentiation efficiency is one of the major drawbacks of these approaches. Here, we compared the efficiency of two methods of neuronal differentiation from iPSCs cultured in two different culture media, StemFlex Medium (SFM) and Essential 8 Medium (E8M). The results indicated that iPSCs cultured in E8M efficiently generated different types of neurons in a shorter time and without the growth of undifferentiated non-neuronal cells in the culture as compared to those generated from iPSCs in SFM. Furthermore, these neurons were validated as functional units immunocytochemically by confirming the expression of mature neuronal markers (i.e., NeuN, Beta tubulin, and Synapsin I), and whole-cell patch-clamp recordings. Long-read single-cell RNA sequencing confirms the presence of upper and deep layer cortical layer excitatory and inhibitory neuronal subtypes in addition to small populations of GABAergic neurons in day 30 neuronal cultures. Pathway analysis indicated that our protocol triggers the signaling transcriptional networks important for the process of neuronal differentiation in vivo.

Cell-free chromatin particles released from dying cancer cells activate immune checkpoints in human lymphocytes: implications for cancer therapy.

Immune checkpoint blockade is the exciting breakthrough in cancer, but how immune checkpoints are activated is unknown. We have earlier reported that cell-free chromatin particles (cfChPs) that circulate in blood of cancer patients, or those that are released locally from dying cancer cells, are readily internalized by healthy cells with biological consequences. Here we report that treatment of human lymphocytes with cfChPs isolated from sera of cancer patients led to marked activation of the immune checkpoints PD-1, CTLA-4, LAG-3, NKG2A, and TIM-3. This finding was corroborated in vivo in splenocytes of mice when cfChPs were injected intravenously. Significant upregulation of immune checkpoint was also observed when isolated lymphocytes were exposed to conditioned medium containing cfChPs released from hypoxia-induced dying HeLa cells. Immune checkpoint activation could be down-regulated by pre-treating the conditioned media with three different cfChPs deactivating agents. Down-regulation of immune checkpoints by cfChPs deactivating agents may herald a novel form of immunotherapy of cancer.