Implantation and integration of human neural stem cells (hNSCs) to mouse host brains has been reported by previous studies. We are interested in generating a pre-organised construct from hNSCs that contains progenitors with different cerebral cortical layer identity using 3D printing technologies (Jin et al., Nat Commun 14, 5986 (2023). We optimised a sequential isolation protocol of lower and upper cortical layer specific hNSCs generated from human pluripotent stem cells (hPSCs). We confirmed the ability of these cells to generate neurons of the upper or lower cortical layers both in vitro and in vivo. We used microfluidic technology to create fused 3D constructs with two compartments that contained early or late NSCs that produced simplified cortical columns. When implanted in immunocompromised postnatal mice (P8), the constructs grew and integrated with the mouse brain and developed connections to distant targets. The implanted constructs became populated by blood vessels, microglia, and astrocytes as early as 2 weeks post grafting and this further increased by 2 months. We showed that hNPCs in monolayer cultures have greater survival and generate more neurons when co-cultured with murine astrocytes. When hNPCs printed together with astrocytes, constructs survived better in vivo, and enhanced integration of the neurons to the host brain. This research will advance basic knowledge of human cortical development with added potential for translational medicine.