Cell Transplantation

End-stage organ failure is a major cause of death worldwide that can occur in patients of all ages and transplantation is the current standard of care for chronic end-stage disease of many organs. Despite the success of organ transplantation, it is becoming clear that there will never be enough organs made available through donation to meet the increasing demand. The past decade's rapid advancement in stem cell biology and tissue engineering generated an explosive outburst of reports that gave rise to regenerative medicine, a new field that promises to “fix” damaged organs through regeneration provided by transplanted cells, stimulation of endogenous repair mechanisms, or implantation of bioengineered tissue. Whether, and if so when, regenerative medicine will keep its promise is uncertain. As we continue to strive to find new effective solutions, alternative approaches based on the development of targeted, preventive interventions aimed at maintaining normal organ function, instead of repairing organ damage, should also be pursued.

Researchers face many challenges, both scientific and societal, in the field of tissue engineering. Herein we discuss the challenges in material design, selection of therapeutic cell source, the in vitro culturing of cells and materials, and finally the integration of the cultured construct into the body. We focus special attention on a new approach to the design of a biomaterial that would bridge synthetic and biologic materials seamlessly. The scaffolds we have developed serve as a transitional material between biotic and abiotic systems.

Our objective in this work was to develop a flexible, biodegradable scaffold for cell transplantation that would incorporate a synthetic component for strength and flexibility and type I collagen for enzymatic lability and cytocompatibility. A biodegradable poly(ester urethane)urea was synthesized from poly(caprolactone), 1,4-diisocyanatobutane, and putrescine. Using a thermally induced phase separation process, porous scaffolds were created from a mixture containing this polyurethane and 0%, 10%, 20%, or 30% type I collagen. The resulting scaffolds were found to have open, interconnected pores (from 7 to >100 um) and porosities from 58% to 86% depending on the polyurethane/collagen ratio. The scaffolds were also flexible with breaking strains of 82–443% and tensile strengths of 0.97–4.11 MPa depending on preparation conditions. Scaffold degradation was significantly increased when collagenase was introduced into an incubating buffer in a manner that was dependent on the mass fraction of collagen present in the scaffold. Mass losses could be varied from 15% to 59% over 8 weeks. When culturing umbilical artery smooth muscle cells on these scaffolds higher cell numbers were observed over a 4-week culture period in scaffolds containing collagen. In summary, a strong and flexible scaffold system has been developed that can degrade by both hydrolysis and collagenase degradation pathways, as well as support cell growth. This scaffold possesses properties that would make it attractive for future use in soft tissue applications where such mechanical and biological features would be advantageous.

Regenerative medicine approaches for the treatment of damaged or missing myocardial tissue include cell-based therapies, scaffold-based therapies, and/or the use of specific growth factors and cytokines. The present study evaluated the ability of extracellular matrix (ECM) derived from porcine urinary bladder to serve as an inductive scaffold for myocardial repair. ECM scaffolds have been shown to support constructive remodeling of other tissue types including the lower urinary tract, the dermis, the esophagus, and dura mater by mechanisms that include the recruitment of bone marrow-derived progenitor cells, angiogenesis, and the generation of bioactive molecules that result from degradation of the ECM. ECM derived from the urinary bladder matrix, identified as UBM, was configured as a single layer sheet and used as a biologic scaffold for a surgically created 2 cm2 full-thickness defect in the right ventricular free wall. Sixteen dogs were divided into two equal groups of eight each. The defect in one group was repaired with a UBM scaffold and the defect in the second group was repaired with a Dacron patch. Each group was divided into two equal subgroups (n = 4), one of which was sacrificed 15 min after surgical repair and the other of which was sacrificed after 8 weeks. Global right ventricular contractility was similar in all four subgroups groups at the time of sacrifice. However, 8 weeks after implantation the UBM-treated defect area showed significantly greater (p < 0.05) regional systolic contraction compared to the myocardial defects repaired with by Dacron (3.3 ± 1.3% vs. −1.8 ± 1.1%; respectively). Unlike the Dacron-repaired region, the UBM-repaired region showed an increase in systolic contraction over the 8-week implantation period (–4.2 ± 1.7% at the time of implantation vs. 3.3 ± 1.3% at 8 weeks). Histological analysis showed the expected fibrotic reaction surrounding the embedded Dacron material with no evidence for myocardial regeneration. Histologic examination of the UBM scaffold site showed cardiomyocytes accounting for approximately 30% of the remodeled tissue. The cardiomyocytes were arranged in an apparently randomly dispersed pattern throughout the entire tissue specimen and stained positive for α-sarcomeric actinin and Connexin 43. The thickness of the UBM graft site increased greatly from the time of implantation to the 8-week sacrifice time point when it was approximately the thickness of the normal right ventricular wall. Histologic examination suggested complete degradation of the originally implanted ECM scaffold and replacement by host tissues. We conclude that UBM facilitates a constructive remodeling of myocardial tissue when used as replacement scaffold for excisional defects.

The potential for endogenous or supplementary stem cells to restore the form and function of damaged tissues is particularly promising for overcoming the restricted regenerative capacity of the mammalian heart. To maintain blood circulation, this essential organ needs to launch a rapid response to repair damage of the muscle wall and to prevent muscle loss. The capacity of growth factors to supplement the repair process has been successfully applied to restore the integrity of damaged skeletal muscle, reducing the fibrotic response to injury, and recruiting local populations of self-renewing precursor cells and circulating stem cells. We review the recent evidence that extension of growth factor supplementation to the heart may overcome its inherent regenerative impediments through improvement of the local tissue environment and stimulation of cell replacement, and we speculate on future research directions for treatment of myocardial damage.

Despite improvements in emergency treatment, myocardial infarction is often the beginning of a downward spiral leading to congestive heart failure. Other than heart transplantation, current therapeutic means aim at enabling the organism to survive with a heart that is working at a fraction of its original capacity. It is therefore no surprise that cardiac stem cell therapy has raised many hopes. However, neither the ideal source and type of stem cell nor the critical cell number and mode of application have been defined so far. Early reports on myocardial repair by adult bone marrow stem cells from rodent models promoted an unparalleled boost of clinical and experimental cell therapy studies. The phenomenon of stem/progenitor cell-induced angiogenesis in ischemic myocardium has ever since been reproduced by numerous groups in a variety of small and large animal models. Myogenesis, however, is an altogether different matter. Many of the initial clinical studies were fueled by the suggestion that early hematopoietic stem cells have a plasticity high enough to enable cross-lineage differentiation into cells of cardiomyocyte phenotype, but the initial enthusiasm has largely faded. The myogenic potential of stroma cell-derived mesenchymal stem cells is much better documented in animal models, but transfer to the clinical setting faces a variety of obstacles. In clinical pilot trials, we and others have demonstrated the feasibility and safety of administering progenitor cells derived from autologous bone marrow to the myocardium of patients with ischemic heart disease. Clinical efficacy data are still rare, but the few controlled trials that have been completed uniformly show a tendency towards better heart function in cell-treated patients. This review is an attempt to describe the scientific basis for cardiac cell therapy from the point of view of the clinician, focusing on problems that arise with beginning translation into the clinical setting.

An increasing need for liver transplantation requires evaluation and triage of organs harvested from “extended criteria” donors. Although there is currently no widely accepted definition, most would agree that “extended criteria” includes organs donated by individuals that are old (>65 years), obese, infected with HBV or HCV, non-heart beating (NHBD), or had an unstable blood pressure before harvesting or the organ experienced a long cold ischemic time. These organs carry a statistical risk of dysfunction early after transplantation, but in the majority of recipients, hepatic parenchymal function recovers. Later, however, a small but significant percentage of extended criteria donors develop biliary strictures within several months after transplantation. The strictures occur primarily because of preservation injury that leads to “ischemic cholangitis” or deep wounding of the bile duct wall. Subsequent partial wound healing and wound contraction, but failed restitution of the biliary epithelial cell (BEC) lining, result in biliary tract strictures that cause progressive biliary fibrosis, increased morbidity, and decreased organ half-life. Better understanding of the pathophysiologic mechanisms that lead to biliary strictures in extended criteria donors provides an ideal proving ground for regenerative medicine; it also can provide insights into other diseases, such as extrahepatic biliary atresia and primary sclerosing cholangitis, that likely share certain pathogenic mechanisms. Possible points of therapeutic intervention include limiting cold and warm ischemic times, donor and/or donor organ treatment, ex vivo, to minimize the ischemic/preservation injury, maximize blood flow after transplantation, promote BEC wound healing, and limit myofibroblasts activation and proliferation in the bile duct wall. The pathobiology of biliary wound healing and therapeutic potential of interleukin-6 (IL-6) are highlighted.

The availability of new cell culture, cell transplantation, and gene expression technology has highlighted a role of hepatocytes as facultative stem cells for the biliary epithelium. In addition, hepatocytes can revert to primitive progenitor cell types and express gene profiles that appear as characteristic “signatures” in hepatocellular carcinomas.

The very limited options available to treat ventricular failure in children with congenital and acquired heart diseases have motivated the development of a pediatric ventricular assist device at the University of Pittsburgh (UoP) and University of Pittsburgh Medical Center (UPMC). Our effort involves a consortium consisting of UoP, Children's Hospital of Pittsburgh (CHP), Carnegie Mellon University, World Heart Corporation, and LaunchPoint Technologies, Inc. The overall aim of our program is to develop a highly reliable, biocompatible ventricular assist device (VAD) for chronic support (6 months) of the unique and high-risk population of children between 3 and 15 kg (patients from birth to 2 years of age). The innovative pediatric ventricular assist device we are developing is based on a miniature mixed flow turbodynamic pump featuring magnetic levitation, to assure minimal blood trauma and risk of thrombosis. This review article discusses the limitations of current pediatric cardiac assist treatment options and the work to date by our consortium toward the development of a pediatric VAD.

Type 1 diabetes (T1D) is an autoimmune disease in which the clinical onset most frequently presents in adolescents who are genetically predisposed. There is accumulating evidence that the endocrine pancreas has regenerative properties, that hematopoietic chimerism can abrogate destruction of beta cells in autoimmune diabetes, and that, in this manner, physiologically sufficient endogenous insulin production can be restored in clinically diabetic NOD mice. Recapitulating what also has been seen sporadically in humans, we set out to test reliable and clinically translatable alternatives able to achieve these same goals. Recently, Tian and colleagues demonstrated that T1D can be prevented in genetically susceptible mice by substituting a “diabetes-susceptible” class II MHC beta chain with a “diabetes-resistant” allelic transgene on their hematopoietic stem cells through gene supplantation. The expression of the newly formed diabetes-resistant molecule in the reinfused hematopoietic cells was sufficient to prevent T1D onset even in the presence of the native, diabetogenic molecule. If this approach to obtain autoimmunity abrogation could facilitate a possible recovery of autologous insulin production in diabetic patients, safe induction of an autoimmunity-free status might become a new promising therapy for T1D.

It is widely believed that human embryonic stem (huES) cells may represent a valid alternative to donor pancreata as a source of islets for transplantation. Much is known about the transcription factors whose sequential activation results in the generation of islets during pancreatic development. This knowledge has been used to articulate the theoretical possibility that such process might be recapitulated in vitro from stem cells. However, our understanding of the extracellular signals that prompt the developing pancreas to follow this sequence of molecular events is very limited. Also, the simplicity of in vitro systems makes it difficult, if not impossible, to mimic the complex signaling pattern observed in living embryos. Protein transduction (PT) technology may provide researchers with a new powerful tool to sequentially induce stem cell differentiation, entirely bypassing the need for unraveling the signaling pattern that drives the process in vivo. Here we discuss this novel application of the flourishing PT technology, which may revolutionize the way we direct stem cells along any specific lineage.

Hybrid extracorporeal liver support is an option to assist liver transplantation therapy. An overview on liver cell bioreactors is given and our own development is described. Furthermore, the prospects of the utilization of human liver cells from discarded transplantation organs due to steatosis, cirrhosis, or traumatic injury, and liver progenitor cells are discussed. Our Modular Extracorporeal Liver Support (MELS) concept proposes an integrative approach for the treatment of hepatic failure with appropriate extracorporeal therapy units, tailored to suit the actual clinical needs of each patient. The CellModule is a specific bioreactor (charged actually with primary human liver cells, harvested from human donor livers found to be unsuitable for transplantation). The DetoxModule enables albumin dialysis for the removal of albumin-bound toxins, reducing the biochemical burden of the liver cells and replacing the bile excretion of hepatocytes in the bioreactor. A Dialysis Module for continuous veno-venous hemofiltration can be added to the system if required in hepato-renal syndrome.

Hepatocyte transplantation has been proposed as a method to support patients with liver insufficiency. There are three main areas where the transplantation of isolated hepatocytes has been proposed and used for clinical therapy. Cell transplantation has been used: 1) for temporary metabolic support of patients in end-stage liver failure awaiting whole organ transplantation, 2) as a method to support liver function and facilitate regeneration of the native liver in cases of fulminant hepatic failure, and 3) in a manner similar to gene therapy, as a “cellular therapy” for patients with genetic defects in vital liver functions. We will briefly review the basic research that leads to clinical hepatocyte transplantation, the published clinical experience with this experimental technique, and some possible future uses of hepatocyte transplantation.

Adult stem cells have been characterized in several tissues as a subpopulation of cells able to maintain, generate, and replace terminally differentiated cells in response to physiological cell turnover or tissue injury. Little is known regarding the presence of stem cells in the adult kidney but it is documented that under certain conditions, such as the recovery from acute injury, the kidney can regenerate itself by increasing the proliferation of some resident cells. The origin of these cells is largely undefined; they are often considered to derive from resident renal stem or progenitor cells. Whether these immature cells are a subpopulation preserved from the early stage of nephrogenesis is still a matter of investigation and represents an attractive possibility. Moreover, the contribution of bone marrow-derived stem cells to renal cell turnover and regeneration has been suggested. In mice and humans, there is evidence that extrarenal cells of bone marrow origin take part in tubular epithelium regeneration. Injury to a target organ can be sensed by bone marrow stem cells that migrate to the site of damage, undergo differentiation, and promote structural and functional repair. Recent studies have demonstrated that hematopoietic stem cells were mobilized following ischemia/reperfusion and engrafted the kidney to differentiate into tubular epithelium in the areas of damage. The evidence that mesenchymal stem cells, by virtue of their renoprotective property, restore renal tubular structure and also ameliorate renal function during experimental acute renal failure provides opportunities for therapeutic intervention.

Reconstructive surgery using autologous vessels is the conventional approach for substitution of diseased vessels or for generation of bypass to improve blood supply downstream of stenosed vessels. In some circumstances the use of autologous material is not possible due to concomitant diseases or previous use, and artificial grafts must be used. Unfortunately, these grafts cannot substitute small-caliber arterial vessels because of thrombotic complications. The objective of tissue engineering at the vascular level is then to generate biological substitutes of arterial conduits with functional characteristics of native vessels, combining cellular components with biodegradable scaffolds. These research projects started in several laboratories, in the late 1990s, and have expanded in different directions using a number of experimental approaches. The objective of this review is to give an overview of the results so far obtained in this area of research, and to discuss the problems related to these investigations, at the experimental and clinical level. The article provides an overview of different biodegradable scaffolds used, experimental techniques for vessels maturation in vitro under mechanical stimulation, and of differentiated as well as precursors of vascular cells, which opens new opportunities for further development of this form of cell transplantation. Finally, the current available results in clinical research will be discussed.