Free from the limitations posed by exogenous scaffolds or extracellular matrix-based materials, scaffold-free engineered tissues have immense clinical potential. Biomaterials may produce adverse responses, interfere with cell–cell interaction, or affect the extracellular matrix integrity of cells. The scaffold-free Kenzan method can generate complex tissues using spheroids on an array of needles but could be inefficient in terms of time, as it moves and places only a single spheroid at a time. We aimed to design and construct a novel scaffold-free bioprinter that can print an entire layer of spheroids at once, effectively reducing the printing time. The bioprinter was designed using computer-aided design software and constructed from machined, 3D printed, and commercially available parts. The printing efficiency and the operating precision were examined using Zirconia and alginate beads, which mimic spheroids. In less than a minute, the printer could efficiently pick and transfer the beads to the printing surface and assemble them onto the 4 × 4 needles. The average overlap coefficient between layers was measured and found to be 0.997. As a proof of concept using human induced pluripotent stem cell-derived spheroids, we confirmed the ability of the bioprinter to place cellular spheroids onto the needles efficiently to print an entire layer of tissue. This novel layer-by-layer, scaffold-free bioprinter is efficient and precise in operation and can be easily scaled to print large tissues.

The role of p53 transactivation domain (p53-TAD), a multifunctional and dynamic domain, on DNA repair and retaining DNA integrity in human induced pluripotent stem cells (hiPSCs) has never been studied. p53-TAD was knocked out in iPSCs using CRISPR/Cas9 and was confirmed by DNA sequencing. p53-TAD knockout (KO) cells were characterized by accelerated proliferation, decreased population doubling time, and unaltered Bcl-2, Bcl-2-binding component 3, insulin-like growth factor 1 receptor, and Bax and altered Mdm2, p21, and p53-induced death domain transcript expression. In p53-TAD KO cells, the p53-regulated DNA repair proteins xeroderma pigmentosum group A, DNA polymerase H, and DNA-binding protein 2 expression were found to be reduced compared with p53 wild-type cells. Exposure to a low dose of doxorubicin (Doxo) induced similar DNA damage and DNA damage response (DDR) as measured by RAD50 and MRE11 expression, checkpoint kinase 2 activation, and γH2A.X recruitment at DNA strand breaks in both cell groups, indicating that silence of p53-TAD does not affect the DDR mechanism upstream of p53. After removal of Doxo, p53 wild-type hiPSCs underwent DNA repair, corrected their damaged DNA, and restored DNA integrity. Conversely, p53-TAD KO hiPSCs did not undergo complete DNA repair and failed to restore DNA integrity. More importantly, continuous culture of p53-TAD KO hiPSCs underwent G₂/M cell cycle arrest and expressed the cellular senescent marker p16^INK4a. Our data clearly show that silence of the TAD of p53 did not affect DDR but affected the DNA repair process, implying the crucial role of p53-TAD in maintaining DNA integrity. Therefore, activating p53-TAD domain using small molecules may promote DNA repair and integrity of cells and prevent cellular senescence. NEW & NOTEWORTHY The p53 transactivation domain (TAD) in human induced pluripotent stem cells (iPSCs) was deleted using the CRISPR/Cas9 system. Deletion of p53-TAD resulted in increased cell proliferation but did not affect iPSC pluripotency. DNA repair machinery upstream of p53 was not affected in p53-TAD knockout, but p53-regulated DNA repair proteins xeroderma pigmentosum group A, DNA polymerase H, and DNA-binding protein 2 were affected. Finally, p53-TAD was found to be necessary for iPSCs to repair damaged DNA and retain DNA integrity.

DNA damage accrued in iPS-derived cardiomyocytes (iPS-CMs) during in vitro culture practices lessens their clinical potentials for therapy. We determined whether DNA damage-free iPSCs (DdF-iPS) can be selected using stabilization of p53, transcription factor that promotes apoptosis in DNA damaged cells, and differentiate them into functionally competent DdF-cardiomyocytes (DdF-CM). p53 was activated using Nutlin-3a in iPSCs to selectively kill the DNA damaged cells, and the stable DdF cells were cultured further and differentiated into CM. Both DdF-iPSCs and DdF-CMs were then characterized. We observed a significant decrease in the expression of reactive oxygen species (ROS) and DNA damage in DdF-iPSCs compared to control (Ctrl)-iPSCs. Next-generation RNA sequencing and Ingenuity pathway analysis revealed improved molecular, cellular, and physiological functions in DdF-iPSCs. The differentiated DdF-CMs had a compact beating frequency between 40 to 60 beats per minute accompanied by increased cell surface area. Additionally, DdF-CMs were able to retain the improved molecular, cellular, and physiological functions after differentiation from iPSCs, and interestingly, cardiac development network was prominent compared to Ctrl-CMs. Enhanced expression of various ion channel transcripts in DdF-CMs implies DdF-CMs is of ventricular CMs and matured compared to its counterpart. Our results indicated that DdF-iPSCs could be selected through p53 stabilization using small molecule and differentiated into ventricular DdF-CMS with fine-tuned molecular signatures. These iPS cells derived DdF-CMs shows immense clinical potential in repairing injured myocardium.

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data in all cells, including stem cells, and its integrity is fundamental to its regenerative ability. Stem cells undergo rapid propagation in labs to achieve the necessary numbers for transplantation. Accelerated cell growth leads to the loss of DNA integrity by accumulated metabolites, such as reactive oxygen, carbonyl, and alkylating agents. Transplanting these cells would result in poor engraftment and regeneration of the deteriorating organ. Moreover, transplanting DNA-damaged cells leads to mutations, DNA instability, cellular senescence, and possibly, life-threatening diseases such as cancer. Therefore, there is an immediate need for a quality control method to evaluate the cell's suitability for transplantation. Here, we provide step-by-step protocols for the assessment of the DNA integrity of stem cells prior to cell transplantation.