| Liver transplantation remains the definitive treatment option for end-stage liver dis-eases. However, the surgical complications, chronic rejections, critical shortage of donororgans and high cost of this procedure have sparked tremendous interest in finding newtreatments. Liver tissue engineering and regenative medicine have emerged as alternativetherapies. Within the past decade, most of the major achievements in these fields have in-volved the production of mimic biological microenvironment that provides appropriatesignals for regulating cellular behavior, which however remains exceedingly difficult toachieve using currently available synthetic or natural materials. There has been an in-creasing emphasis on the use of acellular whole-organ matrices, which can be prepared byremoving the cellular components from donor organs in a process referred to as decellula-rization. Decellularization largely preserves the native composition, ultrastructure and macroscopic three-dimensional architecture of the native extracellular matrix (ECM).Numerous studies have shown that an acellular ECM can be seeded with either functionalparenchymal cells or a specific stem/progenitor cell population, providing initial steps inthe development of new approaches.One of the many challenges in this strategy is the optimization of donor organ decel-lularization. Recently, improved decellularization protocols have been established for exvivo organs, such as the heart, kidney, lung and bladder. A comparison of both the struc-tural and biochemical properties of the ECM scaffold as well as the cell-supporting poten-tial provides relatively ideal scaffolds for further investigation. Currently, decellularizedliver bioscaffold (DLB) is obtained using several different strategies. However, the optim-al technique for liver decellularization has not yet been determined.Another challenge is the supply of functional hepatocytes due to the difficulties asso-ciated with obtaining autologous hepatic tissue and maintaining the phenotype of the pri-mary hepatocytes in culture. Increasing evidence suggests that the differentiation of me-senchymal stem cells (MSCs) into hepatocytes is achieved in the appropriate microenvi-ronment. However, the several traditional protocols used to date have had limited success,and these hepatocyte-like cells exhibit only a portion of the markers and functions of pri-mary hepatocytes. Therefore, further investigations are needed to optimize the direct dif-ferentiation protocol and the culture conditions for MSCs to yield mature hepatocyte-likecells that are fully functional. Recently, studies have emphasized that the differentiation ofstem/progenitor cells is lineage restricted by the tissue-specific biomatrix scaffold. Thisstudy investigates whether DLB promotes the hepatic differentiation of MSCs.In the present study, we comprehensively assessed the structural and biochemicalproperties of rat DLB resulting from four different protocols, with the aim to determine arelatively optimized method for the derivation of DLB with the most positive host re-modeling response and cytocompatibility. We hypothesized that the optimized DLB pro-motes the hepatic differentiation of murine MSCs into high yields of mature hepatocytesin vitro. Furthermore, the therapeutic potential and cell derivation of the pre-differentiatedcells in vitro was investigated in vivo following the intravenous administration of the cellsin a model of chronic liver injury.The main finds are as follows: 1. We characterized DLBs treated using four different decellularization methods todetermine the most effective strategy for the derivation of rat DLB. Althought3-[(3-Cholamidopropyl) dimethylammonio] propanesulfonate (CHAPS) proved inefficientfor the decellularization of rat livers, the other three methods, which are primarily basedon sodium dodecyl sulfate, Triton X-100and nonyl phenoxylpolyethoxylethanol (NP-40)combined with enzymes, successfully yielded DLBs with distinct ultrastructure, ECMcomposition, cell-supporting potential in vitro, and remodeling results as well as patternsof macrophage polarization in vivo. The NP-40-based strategy resulted in a relatively op-timized DLB with enhanced cytotoxicity and host remodeling results. Furthermore, thehost remodeling results statistically correlated with the residual DNA, the number of M2macrophages and the M2:M1cell ratio2. For hepatocyte differentiation, bone marrow derived MSCs isolated fromGFP-transgenic C57BL/6mice possessed the basic features of MSCs as demonstrated bycell morphology and flow cytometry. Results of biocompatibility indicated that DLBtreated with NP-40-based protocol promoted significantly better MSCs cell viability andproliferation in dynamic culture with optimal flow rate during a3-week differentiation pe-riod, when compared to the biomatrix scaffold cultured in static or the monolayer staticculture system.3. The dynamic cultured bioscaffold (DCS), either on its own or in combination withhepatic growth factors (GF), induced the lineage-specific differentiation of MSCs into he-patocyte-like cells expressed hepatocyte-specific markers [eg, α-fetoprotein (AFP), albu-min (ALB), cytoketatins (CK7, CK8, CK18, CK19), hepatic-enriched transcription factors,hepatic functional marker genes and metabolic enzymes] at mRNA and protein levels.Most markers were expressed in DCS group earlier than in the control group. The signifi-cantly higher synthetic and metabolic functions [AFP and ALB secretion, Urea production,glycogen storage, the uptake of indocyaine green and low-density lipoprotein] and the ul-trastructural characteristics of the hepatocyte-like cells in the DCS group further demon-strated the important role of the bioscaffold.4. After the systemic transplantation into a mouse model of CCl4-induced liver fibro-sis, when compared with undifferentiated MSCs or MSCs differentiated using GF alone,the pre-differentiated MSCs produced using the bioscaffold method combined with GF in vitro facilitated the survival of the mice, liver restoration and the long-term functional he-patic integration in vivo. Cell engraftment was significantly improved using thepre-differentiated MSCs by the DCS compared to cells induced in the TCF and the undif-ferentiated MSCs. The inactivation of hepatic stellate cells and the repopulation the resi-dential hepatocytes were promoted by the transplantation of MSCs that were wellpre-differentiated in vitro.In summary, we developed a novel NP-40-based decellularization strategy for thesuccessful derivation of rat DLB, which resulted in the improved cytotoxicity in vitro andhost remodeling results in vivo. In addition, we demonstrated that MSCs could be con-verted into functional hepatocyte-like cells through induction using DLB. As comparedwith the2D conventional induction, these hepatocyte-like cells exhibited higher level andmore stable functions that are potentially useful for the treatment of chronic liver damage.The present study indicates that the3D liver biomatrix might have considerable potentialfor cell-based therapy and tissue engineering. |
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