Trachea Engineering Using a Centrifugation Method and Mouse-Induced Pluripotent Stem Cells

Abstract

The outcomes of tracheal transplantation for the treatment of airway stenosis are unsatisfactory. We investigated the feasibility of regeneration of the trachea using a rat decellularized tracheal scaffold and mouse-induced pluripotent stem (iPS) cells for in vivo transplantation. The rat trachea was first decellularized using a detergent/enzymatic treatment method. We successfully established a centrifugation method that can transplant cells onto the luminal surface of the decellularized rat tracheal scaffold circumferentially. Two types of mouse iPS cells were differentiated into definitive endoderm cells and transplanted onto the luminal surface of the decellularized tracheal matrix scaffold using this centrifugation method. For in vivo study, normal rat tracheas, no-cell rat tracheal scaffolds, or rat tracheal scaffolds recellularized with rat tracheal epithelial cells (EGV-4T) were orthotopically transplanted on F344 rats, and rat tracheal scaffolds recellularized with mouse iPS cells were transplanted on F344/NJc1-rnu/rnu rats. Rats transplanted with no-cell scaffolds or scaffolds recellularized with EGV-4T survived for 1 month, although airway stenosis was observed. One of the F344/NJc1-rnu/rnu rats transplanted with rat trachea regenerated using mouse iPS cells survived over 5 weeks. Histological analysis indicated the cause of death was airway stenosis due to colonic cellular proliferation of undifferentiated iPS cells. Re-epithelialization with numerous ciliated epithelial cells was observed in one of the rats transplanted with trachea bioengineered using iPS cells. In this study, we present a simple and efficient tracheal tissue engineering model using a centrifugation method in a small-animal model. Tissue-engineered trachea using decellularized tracheal scaffolds and iPS cells is potentially applicable for tracheal transplantation.

Preclinical and clinical orthotopic transplantation of decellularized/engineered tracheal scaffolds: A systematic literature review

“…Seeking for a satisfactory decellularization grade of the tracheal tissue, a major part of the Authors recurred to miscellaneous approaches where chemical and enzymatic treatments were combined to physical strategies. The enzymes used included the DNase-I, 1 , 19 , 53 – 57 , 60 eventually mixed with the RNase 58 – 60 or trypsin. 18 …”

“…As expected, to balance the reduced detergents concentration, the time of exposure was extended up to 24 h. A further consideration may concern the consequentiality of the different phases: all the Authors proceeded with detergent first, followed by nucleases treatment. 1 , 18 , 19 , 53 , 55 – 60 Differently, Jang et al, 54 after storing the samples in milliQ water (4°C/48 h), incubated the rabbit tracheas in a single solution consisting in 2000 kU DNase-I in 4% SDC+1 mol/lNaCl for 4 h; while Ohno et al 61 distinguished for a decellularization strategy based on nuclease + chelant instead of detergents. Briefly, the tissues were washed with saline containing 40,000 U/L DNase-I + 20 mM MgCl 2 (2 weeks) and then soaked in saline with 2 mM EDTA (2 weeks), followed by washing with saline (1 week).…”

“…Regarding the physical strategies adopted to boost acellular grafts preparation, immersion in active solutions under agitation was combined with other techniques including the impartment of osmotic and heat shock, hydrostatic pressure, up to ultrasonic bath and vacuum recur. Osmotic shock in distilled water was mainly applied prior to begin with decellularization 1 , 19 , 53 – 57 or, eventually, after a first freezing (heat shock). 58 For Villalba-Caloca et al, 18 Zhang et al, 19 Maughan et al, 59 and Sun et al 60 it was an intermediate phase; while, for Zhou et al, 56 immersion in deionized water was both the first step in decellularization and the last step of each cycle, specifically consisting in 4% SDC (25°C/4 h), 50 kU/mL of DNase-I in 1 M NaCl (37°C/3 h).…”

“…Osmotic shock in distilled water was mainly applied prior to begin with decellularization 1 , 19 , 53 – 57 or, eventually, after a first freezing (heat shock). 58 For Villalba-Caloca et al, 18 Zhang et al, 19 Maughan et al, 59 and Sun et al 60 it was an intermediate phase; while, for Zhou et al, 56 immersion in deionized water was both the first step in decellularization and the last step of each cycle, specifically consisting in 4% SDC (25°C/4 h), 50 kU/mL of DNase-I in 1 M NaCl (37°C/3 h). Together with osmotic and temperature shock, other physical processes included constant agitation, 59 high-hydrostatic pressure, 61 ultrasonic bath, 18 and vacuum.…”

“…Whether approaching a pre-implant seeding, several aspects need to be considered including the most adequate cell source to choose (stem cells, differentiated cells), technical difficulties regarding cells isolation and expansion, seeding and culturing methods; the long times that these procedures take are also a significant drawback. Focusing on the different cell populations used for preliminary colonization of the tracheal scaffolds intended for orthotopic implant, experience is reported with: mouse EGV-4T cells; mouse iPS-MEF-Ng-492B-4 or mouse iPS-Hep-FB/Ng/gfp-103C-1 cells 56 ; adipose MSCs 50 , 58 ; bone marrow MSCs 19 also with bone marrow mononuclear cells 51 ; nasal epithelial cells sheets 17 ; endothelial cells induced from differentiated bone marrow cells 60 ; chondrocytes from bone marrow MSCs and epithelial cells 53 ; amniotic MSCs then differentiated toward chondrogenic lineage. 1 The seeding side is also distinguished, with chondrocytes/derived chondrocytes and epithelial cells or MSCs seeded externally or internally, respectively ( Tables 3 – 6 ).…”