Highly efficient genome editing of human hematopoietic stem cells via a nano-silicon-blade delivery approach

Abstract

Recently, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 bacterial immunity system has opened a promising avenue to treat genetic diseases that affects the human hematopoietic stem cells (HSCs). Therefore, finding a highly efficient delivery method capable of modifying the genome in the hard-to-transfect HSCs, combined with the advanced CRISPR-Cas9 system, may meet the challenges for dissecting the hematologic diseases mechanisms and facilitate future clinical applications. Here, we developed an effective HSC-specified delivery microfluidic chip to disrupt the cell membrane transiently by inducing a rapid mechanical deformation that allowed for delivering biomaterials into cytoplasm from the surrounding matrix. Compared with the previous designs, the new nano-silicon-blade structure was specifically optimized for HSCs. Using the silicon substrate, the sharpness and rigidity of the nano-blade constriction was largely enhanced to improve the biomaterials delivery efficiency. We achieved highly efficient delivery results by transporting various macro-molecules into the HSCs. Moreover, the treated HSCs possess high viability and maintain inherent pluripotency after the delivery via the Nano-Blade Chip (NB-Chip). Subsequently, we disrupted p42 isoform in C/EBPα on the NB-Chip and induced HSCs into a myeloid proliferation behavior. In conclusion, the NB-Chip provides a harmless, rapid and high-throughput gene editing approach for the HSC study and therapeutics.

Intracellular Delivery by Membrane Disruption: Mechanisms, Strategies, and Concepts

“…Such a strategy would presumably disrupt the plasma membrane in a more localized manner, preferentially permeabilizing one side of the cell. To explore this idea, the Qin lab introduced a device with sharp silicon nanoblades protruding from one side of PDMS microfluidic channels 755 . The protruding edge of the silicon nanoblade was essentially formed a spike of ~200 nm radius, creating a gap of ~2 μm for cell passage.…”

“…The protruding edge of the silicon nanoblade was essentially formed a spike of ~200 nm radius, creating a gap of ~2 μm for cell passage. By optimizing the flow rate and number of nanoblade constrictions, they achieved ∼70% delivery efficiency of 70 kDa dextan with ∼80% cell viability in hard-to-transfect HSCs 755 . Compared to electroporation, the delivery efficiency was the same, however, survival and ability of HSCs to remain pluripotent were claimed to be superior with the nanoblade device.…”

Improved Isolation and Culture of Urine-Derived Stem Cells (USCs) and Enhanced Production of Immune Cells from the USC-Derived Induced Pluripotent Stem Cells

“…CFU assays were performed using StemMACS HSC-CFU Media according to the manufacturer's protocol as described previously (MACS miltenyi Biotec, Bergisch Gladbach, NRW, Germany) [54,55]. Briefly, 1 × 10 5 HPCs differentiated from USC-iPSC-1 were mixed with 3 mL of StemMACS HSC-CFU Media (MACS miltenyi Biotec) by vortexing until a homogenous mixture was obtained and then the tubes were incubated at 25 • C for 10 min.…”

Novel fluid shear‐based dissociation device for improved single cell dissociation of spheroids and cell aggregates

“…25 Although all tested device parameters gave similarly high cell viability (Figure 2a), the 125 mm diameter was chosen as the minimum inner diameter of tubing that would encompass the entire spheroid. This inner diameter potentially helped avoid the more detrimental constriction-based dissociation, 18,23 which may have contributed to the high cell viability achieved in our device.…”

“…To eliminate the user‐dependent variability in manual trituration, some studies have focused on using microfluidic flow devices for spheroid dissociation. These devices generally work by manipulating physical forces to dissociate cell aggregates and take advantage of fluid flow through geometries such as pillars, constricts, or inertial forces . Our FSS dissociator also manipulates physical forces of fluid shear to dissociate spheroids but has the advantage of being more scalable than the aforementioned microfluidic devices.…”