Long-term expansion of directly reprogrammed keratinocyte-like cells and in vitro reconstitution of human skin

Abstract

Background: Human keratinocytes and derived products are crucial for skin repair and regeneration. Despite substantial advances in engineered skin equivalents, their poor availability and immunorejection remain major challenges in skin grafting. Methods: Induced keratinocyte-like cells (iKCs) were directly reprogrammed from human urine cells by retroviral transduction of two lineage-specific transcription factors BMI1 and △NP63α (BN). Expression of keratinocyte stem cell or their differentiation markers were assessed by PCR, immunofluorescence and RNA-Sequencing. Regeneration capacity of iKCs were assessed by reconstitution of a human skin equivalent under air-interface condition. Results: BN-driven iKCs were similar to primary keratinocytes (pKCs) in terms of their morphology, protein expression, differentiation potential, and global gene expression. Moreover, BN-iKCs self-assembled to form stratified skin equivalents in vitro. Conclusions: This study demonstrated an approach to generate human iKCs that could be directly reprogrammed from human somatic cells and extensively expanded in serum-and feeder cell-free systems, which will facilitate their broad applicability in an efficient and patient-specific manner.

Comparative Analysis of Olive-Derived Phenolic Compounds’ Pro-Melanogenesis Effects on B16F10 Cells and Epidermal Human Melanocytes

“…The UGCG, keratin (KRT), late cornified envelope (LCE), and small proline-rich protein (SPRR) families are essential factors for keratinocyte differentiation (Figure 4c,d). Differentiated keratinocytes replenish the upper layer and desquamate the dead cells to renew the skin [62]. These results suggest that the olive-derived phenolic compounds might inhibit melanoma growth by disrupting pathways and the cell cycle in melanoma, and might provide the underlying molecular mechanism of the anti-cancer effects of OL and OC reported in previous studies [63][64][65][66].…”

Direct Lineage Reprogramming for Induced Keratinocyte Stem Cells: A Potential Approach for Skin Repair

“… 23 This could be explained based on the expression of the calcium-sensing receptor (CaSR); keratinocytes that express CaSR respond to calcium stimulation during differentiation. 26 Subsequently, Zheng et al 24 showed that the combination of Bmi1 and DNp63α (BD cocktail) can also generate iKSCs from human urine cells that can be cultured for a long-term and can self-assemble to form stratified skin equivalents in vitro under high extracellular calcium conditions. However, the BD cocktail-mediated reprogramming of fibroblasts is less efficient compared to that of human urine cells, and these fibroblast-derived iKSCs cannot form colonies, suggesting that different induction protocols target specific cell types and differentiation might be influenced by the genetic background.…”

“…However, the BD cocktail-mediated reprogramming of fibroblasts is less efficient compared to that of human urine cells, and these fibroblast-derived iKSCs cannot form colonies, suggesting that different induction protocols target specific cell types and differentiation might be influenced by the genetic background. 24 Kurita et al showed for the first time the application of in situ reprogramming in skin repair of animal model. 25 In mice, transduction using adeno-associated viruses (AAV) carrying DNp63α, grainyhead-like 2 (GRHL2), transcription factor AP-2 alpha (TFAP2a), and c-Myc (DGTM cocktail) can induce the conversion of the mesenchymal cells into skin-like epithelial cells at the site of injury, and these cells could proliferate and accelerate wound healing.…”

“…Bmi-1 is a key epigenetic modifier required in this process as it directly binds to cardiac-specific genes and suppresses their expression. 92 However, Bmi-1 and DNP63α can convert human urine cells into iKSCs, 24 indicating that the functions of H2AK119ub are dependent on the genomic context.…”

Generation of Induced Nephron Progenitor-like Cells from Human Urine-Derived Cells

“…To induce reprogramming of human urine cells into iNPCs, human UCs were infected in various combinations of the retroviruses expressing SIX1, SIX2, OSR1, PAX2, OCT4, SOX2, KLF4, c-MYC, and SLUG. As previously established [57,58], these retroviruses were produced by the human 293-derived retroviral packing cell line transfected with pMXs-based retroviral vectors encoding hOCT3/4 (Plasmid #17217), hSOX2 (Plasmid #17218), hKLF4 (Plasmid #17219), hc-MYC (plasmid #17220) and SLUG using Lipofectamine 2000 (Life Technologies, Waltham, MA, USA), according to the manufacturer's instructions. At 96 h post-transfection, retrovirus containing supernatants were harvested, filtered with a 0.45 µm sterile syringe filter (Millipore, Burlington, VT, USA), and concentrated at 20,000× g for 2 h at 4 • C. Human-derived UCs were seeded in a gelatin-coated 6-well plate and, 24 h later, exposed to the concentrated retrovirus in the presence of 4 µg/mL polybrene (Sigma, St. Louis, MO, USA).…”