New and old technologies for organ replacement

INTRODUCTION

About a decade ago, we considered, largely on theoretical grounds, how new and old technologies such as cloning, genetic engineering, tissue engineering, and cellular transplantation might be used to replace or improve the function of failing organs [1–4]. The severe shortage of human organs for transplantation, which slows the delivery of optimum therapy for some and prevents the delivery of optimum therapy for many more patients, motivated these considerations. With the passage of time and advances in medical care and technology, this shortage has become even worse and hence it would seem at least as appropriate now as in the past to consider the dimensions of the problem and solutions with the potential to address to it.

ANTICIPATING FUTURE DEMAND FOR ORGAN REPLACEMENT

The most thorough evaluation of demand for organ transplants and supply of organs for transplantation were published by Evans [5] 10 years ago. Although such thorough analysis has not been updated, one has the sense that the general conclusions reached then seem applicable today. As Table 1 shows, the aggregate demand for organ transplantation, as measured by the number of patients on waiting lists for transplantation, has certainly increased in the last decade (Table 1). But, this table considers only those individuals actually ‘listed’ for transplants and does not include those who might be listed but are not because providers have not considered transplantation as an a therapeutic option or have considered availability of organs too remote. Further, the aggregate increase in demand obscures more dramatic increases in the demand for some organs and decreases in demand for others. Thus, as Fig. 1, taken from the Annual Report of Organ Procurement and Transplantation Network (OPTN) and Scientific Registry of Transplant Recipients (SRTR) [6▪▪], shows the demand for kidney transplants has nearly doubled in the last decade, whereas the demand for lung transplants has nearly halved. At the same time, as Fig. 1 shows, the number of organs available hence for the most part the number of transplants has not changed (except the availability of living donors) and the use of marginal donors has allowed more kidney transplant to be performed [7]. But, this approach neglects the changes in overall population. Adjustment for the 10% increase in the population of the USA in the last decade has had little impact on the demand but reveals that the number of transplants, adjusted for population, has decreased, except for kidney.

Adjustment for population does not impact on the estimates of the current demand for transplantation and might not have served the analysis of prior demand because such adjustments are eclipsed by the improvements in medical and surgical practice that caused transplantation to evolve from a high-risk experiment to a routine procedure. On the other hand, we believe changes in population, demographics, and epidemiology of disease are central to any consideration of how technologies, the development, testing, approval, and routine application of which can span decades (as in the case of organ transplantation) will meet the needs of society. Indeed, some of our early considerations of this subject were remiss in considering only the existing supply and demand [8].

The PEW Research Center has published projections, which, if not particularly surprising, do help one imagine how demand might change over the next 20–30 years [9]. Not only is the population expected to expand during the next 3 decades, but also the demographic profile of the USA will change dramatically [9]. One important change will be in the numbers: the ‘elderly’, defined as those over age 65. In 2030, the elderly will comprise nearly 20% of the population, which will have swelled by nearly 100 million, versus about 12% today. If the next decades bring no other changes, the demand for organ transplantation, which we assume to be cumulative with age, will certainly increase. And, because the incidence and prevalence of heart failure and renal failure increase with age, the demand should increase still more.

Yet another demographic factor could change the demand of transplantation and the supply of organs profoundly. Immigration will account for most if not all of the growth of the population of the USA during the next 30 years [9]. Most immigrants will be Hispanic, some Asian. Immigration could well impact on the types of diseases people experience, the demand for organ transplants, and the frequency of organ donation. Changes in immigration law and extension of health benefits should also increase the demand.

On the other hand, one might hope that advances in medicine and surgery that bring new cures and improvements in care for diseases might in turn decrease the demand for organ transplants. Yet, experience in Europe and the USA suggests otherwise. Thus, greater appreciation of the importance of controlling hypertension and hypercholesterolemia, and the availability of new drugs and devices have dramatically decreased the morbidity and mortality of cardiovascular disease in Europe and the USA [10], yet the number of patients listed for heart transplants has increased [11▪▪]. Nor will changes in the epidemiology of disease likely decrease the demand. As mortality of cardiac disease in the USA decreased from approximately 300 per 100 000 population to approximately 150 per 100 000, the prevalence of diabetes and hypertension increased [12].

Discoveries and advances in molecular diagnosis, genomics, personalized medicine, etc., might increase still further the demand for organ transplantation. Transplantation of the kidney [13], liver [14,15], and pancreas [16] are occasionally performed in patients considered to be at high risk of developing lethal disease such as cancer. Although these procedures are rare today, the demand will only expand as the basic and clinical knowledge increases [17].

NEW AND OLD TECHNOLOGIES FOR REPLACING ORGAN FUNCTION

We previously considered how new and existing technologies might be applied separately and together toward the replacing of organ function [1,4,18]. Some of these technologies are listed in Table 2 and an example of how the technologies might be combined is illustrated in Fig. 2. Ten years ago, one might thoroughly review and critically discuss this subject in a communication such as this one; however, expansion of knowledge and technique makes that all but impossible today. Instead, we shall offer our perspective on a few key advances and unanswered questions.

One subject that has resonated with the scientists, physicians, and the public has been the potential use of stemcells to treat disease, including organ failure [19]. Some applications of stem cells have focused on repairing damaged organs such as the heart [20▪▪]. Others have focused on generating new tissues and organs. We shall consider only the later, and for the most part strategies in which stem cells are coaxed to differentiate into functioning tissue or an organ that can be transplanted into a patient.

Because tissues and organs contain numerous types of cells in more or less complex anatomic arrays, efforts to generate new tissues and organs usually involve the use of stem cells which can proliferate for many generations, perhaps indefinitely and differentiate into many different types of mature cells, that is, pluripotent stem cells [19,21]. Pluripotent stem cells were first isolated from the murine teratomas and after injection into the murine blastocysts were found to contribute to the formation of all tissues in the mosaic offspring [22,23]. The archetype of pluripotent stem cells is the embryonic stem cell [24]. Because embryonic stems are inevitably histoincompatible with those who would need treatment, effective use of these cells and especially of their differentiated progeny seemed to depend on the selection of least incompatible cells from a ‘library’ or engineering of histocompatibility [21]. Still, no matter how large a library of embryonic cells might be and despite the intensive efforts to engineer cells to make them less histoincompatible, one can no more assume embryonic stem cells can be selected or made histocompatible than one can assume bone marrow stem cells or donated organs can be selected or made histocompatible. Hence, the use of these cells for life-sustaining functions would probably require immunosuppression. Use of embryonic stem cells and their progeny is also hindered by the ethical concerns and by the observation in nearly every experimental system that after the transfer into histocompatible and even some histoincompatible individuals, the cells would generate teratomas and teratocarcinomas [25].

Transfer of nuclei into a zygote or blastocyst cell to bring about nuclear reprogramming can achieve better histocompatibility. Nuclear transfer can generate stem cells that are manifestly pluripotent and capable of generating intact animals, and capable of contributing to the germline [26]. Commonly referred to as ‘reproductive cloning’ when newly fashioned cells are used to generate whole animals and ‘therapeutic cloning’ when cells, tissues, or organs are to be produced [1,4,27], nuclear transfer could in principle be used to generate histocompatible cells for efforts to repair or replace failing organs. However, the progeny of stem cells generated by the nuclear transfer are not fully histocompatible with the source of the nucleus because the stem cells has the mitochondrial genome of the reprogramming cell, which encodes some minor histocompatibility antigens and the cells like embryonic stem cells, which generate teratomas [28]. And, if the ‘reprogramming cell’ derives from a human embryo, this approach provokes the same ethical concerns as the use of embryonic stem cells. Some efforts have been made to use xenogeneic embryonic cells to reprogram mature cells, but the full promise of this approach is not yet clear.

Fortunately, the recent years have brought dramatic advances in the understanding and practice of nuclear reprogramming and with it those increasing hope that stem cells might be used to repair or replace failing organs. Nuclear reprogramming can now be accomplished by the expression of a definable set of genes in mature cells [29,30], yielding ‘induced pluripotent stem cells’ or ‘iPS cells’. Expression of these genes is usually accomplished by the transfer of viral transforming genes [30,31▪▪,32▪▪]; however, fusion of donated oocytes with somatic cells [33], exposure to extracts from primitive cells [34], and defined substances might soon replace the need for gene transfer [35▪▪].

Generation of pluripotent stem cells with such ‘cloning factors’ [18], a term we use to denote averting of gene transfer, is especially pertinent to tissue and organ replacement because with the exception of the fusion approach, the genome of the reprogrammed cells, both nuclear and mitochondrial, would derive fully from the donor of the mature cell, that is, the person to be treated. Hence, the stem cell should have no foreign genes. Further, generation of pluripotent stem cells in this way does not require use or destruction of a human embryo and hence avoids the most vexing ethical concerns. Still, induced pluripotent stem cells can form tumors [36] and hence, with full or near histocompatibility, this barrier remains.

Our commentary, to this point, has assumed that pluripotent stem cells containing no foreign DNA would be fully histocompatible with the individual from whom the DNA was obtained. However, some recent work has challenged this concept. Teratomas generated by the inoculation of induced pluripotent stem cells into the mice of the same strain were found to be immunogenic and indeed to be rejected in contrast to the teratomas formed from the embryonic stem cells [37▪▪]. Needless to say, this observation seemed to indicate that induced pluripotent stem cells might not be immunologically superior to embryonic stem cells. However, this unexpected ‘alloimmune’ phenomenon has not been observed by all investigators and indeed it might be peculiar to the experimental system used [35▪▪,38▪▪]. From our perspective, we think the apparent immunogenicity of the induced pluripotent stem cells can be explained by the technical factors and that the key questions will not be immunogenicity, as immunosuppression, if needed, is obviously acceptable to allow organ replacement. Rather, the key questions to us are whether pluripotent stem cells will generate tumors, whether applications using those cells can be made efficient enough to impact on the problem of organ failure, and whether the cells will be able to generate organ replacements. We will consider this third question in the section that follows.

CAN PLURIPOTENT STEM CELLS REPAIR OR REPLACE FAILING ORGANS?

From our perspective, the most daunting challenge key for those who would use stem cells to replace the function of failing organs is whether and how those cells can be parlayed into an organ or organ-like structure of sufficient mass and aggregate function to actually serve the afflicted patient. As teratoma cells [22], embryonic stem cells [39], stem cells cloned by nuclear transfer [26,40], and induced pluripotent stem cells [30,39] can generate whole animals with full-sized, functioning organs, this challenge should be surmountable. Pluripotent stem cells, originated from fibroblasts, have been used to generate enough insulin-secreting cells with sufficient function to reverse hyperglycemia in mice with type 2 [41] and type 1 [42] diabetes. Generation of hepatocyte-like cells from human-induced pluripotent stem cells has been reported [43], but whether cells such as these can be made fully mature and functional has been questioned [44]. As pluripotent stem cells of various types can generate whole animals, we think the means will be found to use them to generate mature and functional cells.

The larger question, in our view, is how to generate an implantable structure that can replace the function of an organ. Even if the replacement of the beta cells of a pancreas can be envisioned and generation of mature hepatocytes to supplement hepatic function is at hand, a whole intact heart, kidney, or lung is not produced. Of course, the heart, and perhaps someday the lung, might be replaced by a device. But, implantation of a living organ or organ-like structure, even if it is not fully compatible with host, will be preferred in some circumstances and hence the question is whether and how generation of an intact, complex organ can be accomplished.

Four approaches for the generation of organ replacements will be discussed briefly here. First, mature tissue and even intact rodent organs have been generated in vitro by exploiting the natural ability of undifferentiated but committed fetal cells to form relatively differentiated and complex structures [45]. Organogenesis in vitro can yield fully differentiated cells [46]; however, tissues so generated lack blood vessels and do not approach the size of fetal organs [47], and achieving the size needed to replace an organ in a mature human would seem beyond the current or anticipated capabilities.

Second, artificial devices and implantable organs have been proposed, and some constructed by seeding matrices, including de-cellularized organs, with mature cells or their precursors [48,49▪▪]. Perhaps, the most advanced of these are the ‘bio-artificial liver devices’, which in experimental systems and clinical trials can exhibit discernable levels of function and clinical benefit [50]; however, the devices have not been advanced to the point where they could be applied for the long-term treatment of hepatic failure [51,52]. Nor have autologous stem cells been used toward this end [53]. Thus, from a current perspective, an implantable, long-term liver replacement stands beyond the current technologies. Efforts are being made to use de-cellularized hearts and kidneys as a substrate for generating implantable cardiac and renal replacements [48,49▪▪]. Seeding de-cellularized rodent heart with cardiac muscle cells and endothelial cells has yielded exciting levels of function in vitro [54], but long-term in-vivo testing and especially the development and testing of hearts repopulated using stem cells or their derivatives have not been reported and would seem remote, at least today.

Third, undifferentiated fetal cells or partly differentiated stem cells or their derivatives might be implanted into the person to be treated, allowing in-vivo organogenesis to occur [55,56]. Primitive cells from the animal fetuses have been advanced with some success; however, use of pluripotent stem cells in lieu of fetal cells has not been tested.

Fourth, stem cells, of various types, might be engrafted into a developing xenogeneic fetus, allowing the microenvironment and growth factors of the fetus to coax the development of mature cells or of an organ, which can be transplanted into the person to be treated [1,4,18]. Human hematopoietic stem cells engrafted in fetal swine generated a functional human immune system in the offspring [57] and some mature human nephrons in the swine kidneys [58]. However, whether this approach could be used to generate primordia in turn capable of generating whole human organs has not been tested and we fear the hurdles to doing so are considerable.

In sum, although many now envision the use of stem cells to generate organs or replace organ functions, no particular approach has emerged to accomplish that end. Still, even if one or several of these approaches prove biologically unfeasible, others mentioned above or yet to be discovered will surely succeed. Rather, the limitation to replacing organ function may eventually reside in the barriers beyond technology.

CONCLUSION

The demand for organ transplantation today far exceeds the supply of human organs. We estimate that this demand, over the next 2–3 decades, which the history of organ transplantation suggests to be the time needed to establish novel technologies, will grow dramatically. And, even with the advances in medicine and public policies, we doubt the supply of human organs will ever keep pace with the demand and hence there will remain an urgency of finding other approaches and this urgency will increase with time. Organs generated in one way or another using the stem cells derived from the individual to be treated seem the most obvious, if not entirely ripe, solution to this problem. Despite the hurdles presently recognized and some yet to be discovered, we have every confidence that safe and reliable means to generate functional human organs or organ replacements will be found. However, we wonder whether society, at least in the USA, will be willing and able to absorb what we think will be a very high cost of generating stem cells for each individual needing treatment, coaxing the stem cells to form organs, and implanting the organs in the vast numbers of an aging population with organ failure, cancer, or other lethal conditions. Generating and testing personalized pluripotent stem cells, expanding the cells to the mass of a kidney or liver, and implanting the personalized organ will be vastly more expensive than allogeneic organ transplantation, even allowing that ongoing immunosuppression may not be needed. We suspect this problem might lead the transplant community either to regain enthusiasm for xenotransplantation or to finally grapple with the ethical challenges of rationing healthcare.