Layer (L)2 is a major output of primary sensory cortex that exhibits very sparse spiking, but the structure of sensory representation in L2 is not well understood. We combined two-photon calcium imaging with deflection of many whiskers to map whisker receptive fields, characterize sparse coding, and quantitatively define the point representation in L2 of mouse somatosensory cortex. Neurons within a column-sized imaging field showed surprisingly heterogeneous, salt-and-pepper tuning to many different whiskers. Single whisker deflection elicited low-probability spikes in highly distributed, shifting neural ensembles spanning multiple cortical columns. Whisker-evoked response probability correlated strongly with spontaneous firing rate, but weakly with tuning properties, indicating a spectrum of inherent responsiveness across pyramidal cells. L2 neurons projecting to motor and secondary somatosensory cortex differed in whisker tuning and responsiveness, and carried different amounts of information about columnar whisker deflection. From these data, we derive a quantitative, fine-scale picture of the distributed point representation in L2.

Huntington disease (HD) is an inherited, progressive neurological disorder characterized by degenerating striatal medium spiny neurons (MSNs). One promising approach for treating HD is cell replacement therapy, where lost cells are replaced by MSN progenitors derived from human pluripotent stem cells (hPSCs). While there has been remarkable progress in generating hPSC-derived MSNs, current production methods rely on two-dimensional culture systems that can include poorly defined components, limit scalability, and yield differing preclinical results. To facilitate clinical translation, here, we generated striatal progenitors from hPSCs within a fully defined and scalable PNIPAAm-PEG three-dimensional (3D) hydrogel. Transplantation of 3D-derived striatal progenitors into a transgenic mouse model of HD slowed disease progression, improved motor coordination, and increased survival. In addition, the transplanted cells developed an MSN-like phenotype and formed synaptic connections with host cells. Our results illustrate the potential of scalable 3D biomaterials for generating striatal progenitors for HD cell therapy.

Oligodendrocyte precursor cells (OPCs) offer considerable potential for the treatment of demyelinating diseases and injuries of the CNS. However, generating large quantities of high-quality OPCs remains a substantial challenge that impedes their therapeutic application. Here, we show that OPCs can be generated from human pluripotent stem cells (hPSCs) in a three-dimensional (3D), scalable, and fully defined thermoresponsive biomaterial system. We used CRISPR/Cas9 to create a NKX2.2-EGFP human embryonic stem cell reporter line that enabled fine-tuning of early OPC specification and identification of conditions that markedly increased the number of OLIG2⁺ and NKX2.2⁺ cells generated from hPSCs. Transplantation of 50-day-old OPCs into the brains of NOD/SCID mice revealed that progenitors generated in 3D without cell selection or purification subsequently engrafted, migrated, and matured into myelinating oligodendrocytes in vivo. These results demonstrate the potential of harnessing lineage reporter lines to develop 3D platforms for rapid and large-scale production of OPCs.