First Application of a Mixed Porcine-Human Repopulated Bioengineered Liver in a Preclinical Model of Post-Resection Liver Failure.

In this study, a mixed porcine-human bioengineered liver (MPH-BEL) was used in a preclinical setup of extracorporeal liver support devices as a treatment for a model of post-resection liver failure (PRLF). The potential for human clinical application is further illustrated by comparing the functional capacity of MPH-BEL grafts as assessed using this porcine PRLF model with fully human (FH-BEL) grafts which were perfused and assessed in vitro. BEL grafts were produced by reseeding liver scaffolds with HUVEC and primary porcine hepatocytes (MPH-BEL) or primary human hepatocytes (FH-BEL). PRLF was induced by performing an 85% liver resection in domestic white pigs and randomized into the following three groups 24 h after resection: standard medical therapy (SMT) alone, SMT + extracorporeal circuit (ECC), and SMT + MPH-BEL. The detoxification and metabolic functions of the MPH-BEL grafts were compared to FH-BEL grafts which were perfused in vitro. During the 24 h treatment interval, INR values normalized within 18 h in the MPH-BEL therapy group and urea synthesis increased as compared to the SMT and SMT + ECC control groups. The MPH-BEL treatment was associated with more rapid decline in hematocrit and platelet count compared to both control groups. Histological analysis demonstrated platelet sequestration in the MPH-BEL grafts, possibly related to immune activation. Significantly higher rates of ammonia clearance and metabolic function were observed in the FH-BEL grafts perfused in vitro than in the MPH-BEL grafts. The MPH-BEL treatment was associated with improved markers of liver function in PRLF. Further improvement in liver function in the BEL grafts was observed by seeding the biomatrix with human hepatocytes. Methods to reduce platelet sequestration within BEL grafts is an area of ongoing research.

Re-Endothelialization of Decellularized Liver Scaffolds: A Step for Bioengineered Liver Transplantation.

Bioengineered livers (BELs) are an attractive therapeutic alternative to address the donor organ shortage for liver transplantation. The goal of BELs technology aims at replacement or regeneration of the native human liver. A variety of approaches have been proposed for tissue engineering of transplantable livers; the current review will highlight the decellularization-recellularization approach to BELs. For example, vascular patency and appropriate cell distribution and expansion are critical components in the production of successful BELs. Proper solutions to these components of BELs have challenged its development. Several strategies, such as heparin immobilization, heparin-gelatin, REDV peptide, and anti-CD31 aptamer have been developed to extend the vascular patency of revascularized bioengineered livers (rBELs). Other novel methods have been developed to enhance cell seeding of parenchymal cells and to increase graft functionality during both bench and in vivo perfusion. These enhanced methods have been associated with up to 15 days of survival in large animal (porcine) models of heterotopic transplantation but have not yet permitted extended survival after implantation of BELs in the orthotopic position. This review will highlight both the remaining challenges and the potential for clinical application of functional bioengineered grafts.

Functional characterization of a bioengineered liver after heterotopic implantation in pigs.

Organ bioengineering offers a promising solution to the persistent shortage of donor organs. However, the progression of this technology toward clinical use has been hindered by the challenges of reconstituting a functional vascular network, directing the engraftment of specific functional cell types, and defining appropriate culture conditions to concurrently support the health and phenotypic stability of diverse cell lineages. We previously demonstrated the ability to functionally reendothelialize the vasculature of a clinically scaled decellularized liver scaffold with human umbilical vein endothelial cells (HUVECs) and to sustain continuous perfusion in a large animal recovery model. We now report a method for seeding and engrafting primary porcine hepatocytes into a bioengineered liver (BEL) scaffold previously reendothelialized with HUVECs. The resulting BELs were competent for albumin production, ammonia detoxification and urea synthesis, indicating the presence of a functional hepatocyte compartment. BELs additionally slowed ammonia accumulation during in vivo perfusion in a porcine model of surgically induced acute liver failure. Following explant of the graft, BEL parenchyma showed maintenance of canonical endothelial and hepatocyte markers. Taken together, these results support the feasibility of engineering a clinically scaled functional BEL and establish a platform for optimizing the seeding and engraftment of additional liver specific cells.

Sustained perfusion of revascularized bioengineered livers heterotopically transplanted into immunosuppressed pigs.

Implanted bioengineered livers have not exceeded three days of continuous perfusion. Here we show that decellularized whole porcine livers revascularized with human umbilical vein endothelial cells and implanted heterotopically into immunosuppressed pigs whose spleens had been removed can sustain perfusion for up to 15 days. We identified peak glucose consumption rate as a main predictor of the patency of the revascularized bioengineered livers (rBELs). Heterotopic implantation of rBELs into pigs in the absence of anticoagulation therapy led to sustained perfusion for three days, followed by a pronounced immune responses directed against the human endothelial cells. A 10 day steroid-based immunosuppression protocol and a splenectomy at the time of rBEL implantation reduced the immune responses and resulted in continuous perfusion of the rBELs for over two weeks. We also show that the human endothelial cells in the perfused rBELs colonize the liver sinusoids and express sinusoidal endothelial markers similar to those in normal liver tissue. Revascularized liver scaffolds that can maintain blood perfusion at physiological pressures might eventually help to overcome the chronic shortage of transplantable human livers.

Author Correction: Sustained perfusion of revascularized bioengineered livers heterotopically transplanted into immunosuppressed pigs.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Multipotent adult progenitor cells sustain function of ischemic limbs in mice.

Despite progress in cardiovascular research, a cure for peripheral vascular disease has not been found. We compared the vascularization and tissue regeneration potential of murine and human undifferentiated multipotent adult progenitor cells (mMAPC-U and hMAPC-U), murine MAPC-derived vascular progenitors (mMAPC-VP), and unselected murine BM cells (mBMCs) in mice with moderate limb ischemia, reminiscent of intermittent claudication in human patients. mMAPC-U durably restored blood flow and muscle function and stimulated muscle regeneration, by direct and trophic contribution to vascular and skeletal muscle growth. This was in contrast to mBMCs and mMAPC-VP, which did not affect muscle regeneration and provided only limited and transient improvement. Moreover, mBMCs participated in a sustained inflammatory response in the lower limb, associated with progressive deterioration in muscle function. Importantly, mMAPC-U and hMAPC-U also remedied vascular and muscular deficiency in severe limb ischemia, representative of critical limb ischemia in humans. Thus, unlike BMCs or vascular-committed progenitors, undifferentiated multipotent adult progenitor cells offer the potential to durably repair ischemic damage in peripheral vascular disease patients.

Evaluation of neural plasticity in adult stem cells.

The role of stem cells has long been known in reproductive organs and various tissues including the haematopoietic system and skin. During the last decade, stem cells have also been identified in other organs, including the nervous system, both during development and in post-natal life. More recently, evidence has been presented that stem cells thought to be responsible for the generation of mature differentiated cells of one organ, such as haematopoietic stem cells, may have the ability to also differentiate across lineages and contribute to tissues other than haematopoietic cells, including neuronal tissue, suggesting that easily accessible stem cells sources may one day be useful in the therapy of ischaemic (stroke) and also degenerative diseases of the nervous system. Here, we will evaluate the validity of such claims based on a number of criteria we believe need to be fulfilled to definitively conclude that certain stem cells can give rise to functional neural cells that might be suitable for therapy of neural disorders.

Cytokine-induced differentiation of multipotent adult progenitor cells into functional smooth muscle cells.

Smooth muscle formation and function are critical in development and postnatal life. Hence, studies aimed at better understanding SMC differentiation are of great importance. Here, we report that multipotent adult progenitor cells (MAPCs) isolated from rat, murine, porcine, and human bone marrow demonstrate the potential to differentiate into cells with an SMC-like phenotype and function. TGF-beta1 alone or combined with PDGF-BB in serum-free medium induces a temporally correct expression of transcripts and proteins consistent with smooth muscle development. Furthermore, SMCs derived from MAPCs (MAPC-SMCs) demonstrated functional L-type calcium channels. MAPC-SMCs entrapped in fibrin vascular molds became circumferentially aligned and generated force in response to KCl, the L-type channel opener FPL64176, or the SMC agonists 5-HT and ET-1, and exhibited complete relaxation in response to the Rho-kinase inhibitor Y-27632. Cyclic distention (5% circumferential strain) for 3 weeks increased responses by 2- to 3-fold, consistent with what occurred in neonatal SMCs. These results provide evidence that MAPC-SMCs are phenotypically and functionally similar to neonatal SMCs and that the in vitro MAPC-SMC differentiation system may be an ideal model for the study of SMC development. Moreover, MAPC-SMCs may lend themselves to tissue engineering applications.

Differentiation of multipotent adult progenitor cells into functional endothelial and smooth muscle cells.

Stem cells are not only a promising in vivo tool for the treatment of diseases characterized by irreversible tissue damage, but can also be exploited as in vitro systems to study the conditions required to generate molecularly and functionally defined cell types. Constructing functional arteries with luminal arterial endothelial cells stabilized by a medial layer of smooth muscle cells is one of the challenges of regenerative medicine. This unit describes the conditions for generating endothelial and smooth muscle cells from multipotent adult progenitor cells (MAPCs). It elaborates on the importance of certain parameters, e.g., quality control of the stem cell population used, serum lot variations, seeding density, use of appropriate cytokines, critical to obtaining high differentiation efficiencies. It further focuses on the molecular and functional characterization of the obtained cell types.