Langenbecks Arch Surg (2009) 394:985997 DOI 10.

1007/s00423-009-0546-0

OVERVIEW

The possible use of stem cells in regenerative medicine: dream or reality?

Sabrina Ehnert & Matthias Glanemann & Andreas Schmitt & Stephan Vogt & Naama Shanny & Natascha C. Nussler & Ulrich Stckle & Andreas Nussler

Received: 13 July 2009 / Accepted: 14 July 2009 / Published online: 31 July 2009 # Springer-Verlag 2009

Abstract Stem cells are one of the most fascinating areas in regenerative medicine today. They play a crucial role in the development and regeneration of human life and are defined as cells that continuously reproduce themselves while maintaining the ability to differentiate into various cell types. Stem cells are found at all developmental stages, from embryonic stem cells that differentiate into all cell types found in the human body to adult stem cells that are responsible for tissue regeneration. The general opinion postulates that clinical therapies based on the properties of stem cells may have the potential to change the treatment of degenerative diseases or important traumatic injuries in the near future. We here briefly review the literature in particularly for the liver, heart, kidney, cartilage, and bone regeneration.
S. Ehnert : A. Schmitt : U. Stckle : A. Nussler (*) Department of Traumatology, TU Munich, Klinikum rechts der Isar, Ismaningerstr. 22, 81675 Munich, Germany e-mail: nuessler@uchir.me.tum.de M. Glanemann : N. Shanny Department of General, Visceral and Transplantation Surgery, Charit, Campus Virchow, Augustenburger Platz 1, 13353 Berlin, Germany S. Vogt Department of Sport Orthopedics, TU Munich, Klinikum rechts der Isar, Ismaningerstr. 22, 81675 Munich, Germany N. C. Nussler Department of General Surgery, Klinikum Neuperlach, Oskar-Maria-Graf-Ring 51, 81737 Munich, Germany

Keywords Embryonic stem cells . Adult stem cells . Liver . Bone . Heart . Kidney

Introduction For many reasons, stem cells are important for living organisms. In the 3- to 5-day-old embryo, the so called blastocyst, stem cells give rise to the multiple specialized cell types that make up the heart, bone, lung, skin, and other tissues. In some adult tissues, such as bone marrow, liver, muscle, and brain, small populations of adult (or somatic) stem cells control replacement of cells which are lost through age, injury, or disease. There are three important characteristics that distinguish stem cells from other cell types. Stem cells are unspecialized but pluripotent (can differentiate into all three major tissue types) cells, which have the ability to indefinitely renew themselves. Scientists put great effort in the understanding, identification, and characterization of the various differentiation processes stem cells may undergo. This leads to an increasing knowledge about how an organism develops from a single cell and how damaged cells are replaced in adult organisms. This opens doors for scientists to investigate the possibility of cell-based therapies to treat disease, which is often referred to as regenerative or reparative medicine. Cell therapy, including the disciplines of regenerative medicine, tissue-, and bio-engineering, is dependent on cell and tissue culture methodologies to generate and expand specific cells in order to replace important differentiated functions lost or altered in various disease states (e.g., no insulin production in diabetes). Central to the successful development of cellbased therapies is the question of cell sourcing. Thus, advances in stem cell research have a vital impact on this problem.

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Scientists primarily work with two kinds of stem cells from animals and humans, namely embryonic stem cells (ESC) and adult (or somatic) stem cells. The history of research on adult stem cells began about 40 years ago. In the 1960s, researchers discovered that the bone marrow contains at least two kinds of stem cells. One population, called hematopoietic stem cells, forms all the types of blood cells in the body. A second population, called bone marrow stromal cells, was discovered a few years later. They are a mixed cell population that generates bone, cartilage, fat, and fibrous connective tissue. More than 20 years ago, scientists discovered ways to obtain stem cells from early mouse embryos. Many years of research with mouse embryonic stem cells were necessary to successfully isolate and grow human embryonic stem cells, for the first time in 1998 [1]. Since then, several human ESC lines have been generated from the inner cell mass of the blastocyst. The embryos used for these cell lines were created by in vitro fertilization. When no longer needed for infertility purposes, they were donated for research with the informed consent of the donor. A registry of them is published on the NIH web site (http://stemcells.nih.gov/registry/). Usage of human ESCs as resource for cell therapeutic approaches is presently an intensive field of research [2]. From a legal and ethical point of view, research involving human embryonic cells is highly controversial, and many countries are reviewing their legislation. The main ethical issues raised are the generation of human ESCs from in vitro fertilized embryos, the moral status of the embryo, and the acceptability of using such derived cells for therapeutic purposes. There are less ethical issues when using adult stem cells. And adult stem cells have further advantages, e.g., their possible use for autologous cell therapies, using the patients own cells to reduce possible immune responses. But there are also limitations of adult stem cells, which typically generate the cell types of the tissue in which they reside. Until recently, it had been thought that a bloodforming adult stem cell in the bone marrow, called hematopoietic stem cell, can only give rise to the many types of blood cells such as red blood cells, white blood cells, and platelets, but not to cells of a very different tissue, such as nerve cells in the brain. However, a number of experiments over the last several years have raised the possibility that stem cells from one tissue may be able to give rise to cell types of a completely different tissue, a phenomenon known as plasticity, offering new perspectives for cell-based therapies. Examples of such plasticity include blood cells becoming neurons [3] or insulin-producing liver cells [4, 5] and hematopoietic stem cells that can develop into heart muscle. Despite these studies, no adult stem cell has been definitively shown to be completely pluripotent. Thus, we have to clearly differentiate between totipotent

(latin totus = entire) stem cell, which can give rise to all the cell types that make up the body and in addition all of the cell types that make up the extra-embryonic tissues (e.g. the placenta); pluripotent stem cells, which give rise to all cell types that make up the body, but not the extra-embryonic tissues; and multipotent stem cells, which can develop into more than one cell type of the body (Fig. 1). A limitation is tried by the researchers to overcome by the generation of so-called induced pluripotent stem cells (iPSC). Per definition, iPSCs are adult cells reprogrammed to an ESC-like state, which express stem-cell markers and are capable of generating cells characteristic of all three germ layers. In 2006, Takahashi and coworkers first reported about the induction of pluripotent stem cells from mouse embryonic fibroblast cultures by expression of transcription factors Oct4, Sox2, c-Myc, and Klf4 [6], which had a similar morphology, proliferation, and teratoma formation than ESCs. Only 1 year later, a major breakthrough was achieved by creating the first iPSCs from adult human cells by retroviral infection with Oct4, Sox2, c-Myc, and Klf4 [7] or Oct4, Sox2, Nanog, and Lin28 [8]. Since then, several groups tried to generate iPSCs from various types of cells, e.g., mouse and human (embryonic) fibroblasts [9], adult dermal fibroblasts [10, 11], neural stem cells [12], and human keratinocytes [13], by retroviral gene-transfer of up to four different factors. Li and coworkers proposed the use of chemical inhibitors to support the genetic reprogramming [14]. Having similar characteristics than ESCs, iPSCs might have a promising future in cell therapy. Although iPSCs raise less ethical questions than ESCs, there are still many points that have to be investigated before these cells can be used for medicine, e.g., cancerogeneicity and safety of retroviral gene transfer.

Application of stem cells in regenerative medicine Two different strategies are pursued for application of stem cells in regenerative medicine. The one and easier way is simple injection of cell suspension into the blood circulation or the target tissue. In contrast to that strategy termed cell therapy, the second approach called tissue engineering is more complex. Here, a tissue-like construct consisting of a three-dimensional matrix colonized by cells is engineered in vitro and subsequently implanted into the tissue defect of the recipient. Mostly, addition of growth factors is necessary for proliferation and differentiation of stem cells in vitro (Fig. 2). The application rate for the cell therapy is mainly the recovery of organ malfunction by substitution of defect cells. One important example for employment of cell therapy is the transplantation of hematopoietic stem cells. This procedure has been practiced successfully for decades

Langenbecks Arch Surg (2009) 394:985997 Fig. 1 Use of different stem cells for research and clinical application
Adult Stem Cells (e.g., liver, heart, bone marrow)

987

Embryonic Stem Cells

Pluripotent Stem Cells

Multipotent Stem Cells

Differentiation in specified cell types

Ectodermal cells

Endodermal Cells

Mesodermal Cells

e.g., Neuron

e.g., Islets or Hepatocytes

e.g., Muscle

Clinical Application:
Basic research, Preclinic or drug Development (efficacy & toxicity)
e.g., Burn diseases, Bone regeneration, organ Regeneration, heart diseases, Neuronal diseases

to treat serious hematological diseases. In the transplantation of bone marrow, hematopoietic stem cells are injected into the blood circulation of the recipient. They find their way to the bone marrow by a phenomenon termed

homing. Chemokines are reported to play a key role in homing of hematopoietic stem cells [15, 16]. A number of experiments have indicated the ability of homing to injured tissue for mesenchymal and embryonic

Cell therapy

Tissue engineering

Fig. 2 Different strategies of regenerative medicine employing stem cells. For cell therapy and tissue engineering stem cells are isolated either from the patient (autologous transplantation) or from other humans (allogene transplantation). For cell therapy, the cells are expanded in vitro and applied directly to the patient to substitute

lost cells, while in case of tissue engineering after expansion, the cells are seeded into three-dimensional scaffolds. There cells are differentiated into the demanded cell type. The obtained artificial tissue construct is implanted into patients tissue defect, where vascularization occurs

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stem cells. After systemic application in animal models of hepatic intoxication and partial hepatectomy, myocardial infarction, nephropathy, cerebral ischemia, lung injury, lung fibrosis, and local irradiation, stem cells are enriched in injured tissue and partially differentiated into tissue-specific cell types [2, 1726]. There have also been systemic applications of mesenchymal stem cells in humans with beneficial effect in graft-versus-host disease or osteogenesis imperfecta [27, 28]. However, cell therapy alone cannot suffice to regenerate large tissue defects or even replace whole organs. Here, the approach of tissue engineering seems to be a promising strategy in future. Thereby, a scaffold imitating the architecture of tissue-specific extracellular matrix is constructed and seeded with tissue-specific cells. The major problem in construction of solid tissue is the supply of cells with oxygen and nutrition. To avoid necrosis, vascularization of the newly constructed tissue is essential. Despite a number of experiments employing co-culture of vascular cells, preforming of vessels in scaffolds, or treatment with pro-angiogenetic cytokines, the problem of vascularization is not yet solved satisfactorily. Tissue engineering was already successfully used to synthesize experimental replacement for simple assembled tissue as skin, bone, or fat [2931]. Anyways, today, tissue engineering is still far from successful generation of tissue from highly complicated architecture as kidney, neural tissue, or intestine for therapeutically replacement.

interconnected pores of a diameter of at least 100m to allow in-growth of cells and vessels [32]. Despite the tissue engineering of bone, where various inorganic materials, such as hydroxyapatite, calcium phosphate, calcium carbonate, or glasses were tested, mainly organic biomaterials have been investigated for scaffold formation. These are either naturally derived as collagen, fibrin, agarose, alginate, gelatin, silk or hyaluronic acid, or synthetically produced. Synthetically produced organic biomaterials are mainly polyhydroxyacids as polyglycolide or polylactide. To control kinetics of degradation recent studies were performed employing hydroxyacid copolymers. Thus, it is tried to adapt kinetics of degradation to those of tissue regeneration. As these synthetic polymers often lack bioactivity, in recent studies, they were surface-modified to alter cell adhesion, migration, differentiation, and proliferation. Thereby, they were coated or copolymerized with bioactive materials, or functional groups were attached to the polymer chain before scaffold fabrication [33, 34]. Besides surface modifications with bioactive materials, scaffolds were coated directly with cytokines to control proliferation and differentiation of seeded cells [35]. Other authors describe coating of scaffolds with gen vectors to perform transfection of cells with different growth factors [36]. Biomaterials for tissue engineering can also carry drugs that prevent microbial colonization or control in-growth of scaffolds into the surrounding tissue [37, 38].

Biomaterials in regenerative medicine Biomaterials are frequently used in regenerative medicine. They act as artificial extracellular matrix with distinct mechanical and biological properties or as carriers for drugs and bioactive molecules. In recent years, a variety of different biomaterials were tested for their capability as matrix in tissue engineering. For this use, materials have to fulfill some fundamental requirements. They have to be biodegradable to allow replacement by regenerated tissue; they must be immunecompatible; and they must neither be toxic nor release toxic substances when they are degraded. Beside these qualities, matrixes formed from biomaterials must have distinct properties with regard to the desired kind of tissue. The request on mechanical strength, kinetics of degradation, and bioactivity significantly varies between different tissues. For example, bone formation requires scaffolds from high mechanical resistance and slow absorption, as regeneration of skin demands soft matrix with fast degradation kinetics. Besides used biomaterials themselves, the three-dimensional structures of scaffolds have great influence on cell growth and differentiation. Scaffolds must be highly porous with

Potential benefits of stem cell therapies Today, the maybe best-known and well-established stem cell therapy is the bone marrow transplantation. In fact, adult blood-forming stem cells from bone marrow have been used in transplants to treat various blood disorders, leukemia, and other types of cancer for 30 years. However, a brief look into the literature will clearly demonstrate that pluripotent and multipotent stem cells are believed to have great potential for both clinical and research applications. Stem cells could be stimulated to develop into specialized cells that represent renewable sources of cells, tissue, or even organs for transplantation. Cell-replacement therapy might be used to treat injuries as well as a great variety of degenerative and genetic disorders. There is a long list of diseases that is believed to be treated by stem cell therapy including several liver diseases, diabetes, muscular dystrophies, Alzheimer disease, Parkinson's disease, arthritis, retinal degeneration, spinal cord injuries, multiple scleroses, and monogenetic disorders such as hemophilia. Established stem cell therapies today include transplantation of bone marrow stem cells (adult stem cell), peripheral blood stem cells (adult stem cell), and umbilical

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cord blood stem cell. But also, many other possibilities for stem cell therapy have been investigated in the past few years. As an example, in patients suffering from type I diabetes, the cells of the pancreas that normally produce insulin are destroyed by the patient's own immune system. New studies indicate that it may be possible to direct the differentiation of human embryonic stem cells in cell culture to form insulin-producing cells that eventually could be used in transplantation therapy for diabetics [3942]. Furthermore, a brief look into Medline shows also the economic potential of stem cells in research. Numerous groups have suggested that stem cells can be used in various aspects of toxicology [43]. It is believed that reproduction toxicology can be controlled by a test recently developed by Axiogenesis in Germany (http://www.axio genesis.com/cms/front_content.php). This company produces cutting-edge mouse embryonic stem cell lines that can be used to generate pure tissue and organ cell types used for drug development and reproduction toxicology. Furthermore, peripheral blood monocytes have been differentiated into hepatocyte-like cells that might be used to test acute and/or chronic toxicology approaches [4, 5, 44].

when several hundred million of native human hepatocytes are instantly available to ameliorate the disease state of a patient. This, however, raises additional questions about availability, cryopreservation, or storage of the cells. But also the route of cell application, engraftment, or homing of cells in the diseased tissue or at least the cell survival in another organ, where they may partially fulfill their initial role as well as the role of immunosuppression, are questions that have been not solved yet. After an initial overview, we like to review the literature on a few potential clinical applications of stem or precursor cells.

Liver The progress made in the field of liver organ transplantation has revolutionized the treatment of a wide spectrum of liver diseases. Nevertheless, cell-based therapies are emerging as an alternative to whole-organ transplantation. There are three categories of liver disease that can be distinguished and principally addressed by liver cell therapy, namely acute liver failure, inherited metabolic, and end-stage liver disease (cirrhosis). We need to emphasize that the conditions and requirements of cell therapy differ for each of these diseases. Thus, hepatocyte transplantation has been used successfully in order to bridge patients to whole organ transplantation [49] to decrease mortality in acute liver failure [50] and to treat metabolic liver disease [51]. Cell-based therapy of end-stage liver disease, however, is more problematic. Besides loss of functional hepatocytes, abnormalities of the hepatic architecture contribute to the decrease in liver function. Intrahepatic portal-to-portal venous shunts may prevent an efficient exchange between hepatocytes and blood plasma. In this situation, the benefit of additionally transplanted hepatocytes into the liver without restoring the normal liver architecture may be questionable. An alternative strategy may be transplantation of hepatocytes into the spleen. To evaluate the efficiency of intrasplenic transplantation several researchers induced liver cirrhosis in rats using phenobarbital or carbon tetrachloride followed by direct injection of cells into the splenic pulp [52, 53]. Only animals with stable liver cirrhosis 4 weeks after the discontinuation of carbon tetrachloride were subjected to cell therapy. With different cell types applied, namely rat or porcine hepatocytes, syngeneic rat hepatocytes or immortalized rat hepatocytes intrasplenic cell therapy clearly improved liver function and prolonged survival [18, 53]. The response to hepatocyte transplantation in humans with end-stage liver disease, however, has not resulted in the same degree of improvement compared to experimental animal studies [54, 55]. One explanation may be that the

Potential risks or problems with future stem cell therapies Besides the overall positive potential of stem cell therapy, it would be extremely thoughtless if we would not discuss at this point at least some risks associated with stem cell therapy. Embryonic stem cells have full developmental potential to become all types of cells or tissues in the body, and they have been reported to integrate into organs such as the kidney [45] or the liver [46]. However, their therapeutic use is limited by the recipients immune responses and the potential development of teratomas [1]. Moreover, the use of human embryos for the production of these cells has raised many ethical issues that remain unsolved. Therefore, its use is quite limited in several countries and has not gained clinical application. In contrary, adult stem cells, particularly hematopoietic and mesenchymal stem cells of the bone marrow, are routinely used in clinical application. They have the great advantage that they can be used for autologous cell therapies. However, a great risk remains particularly in case of mesenchymal stem cells that have been reported to lose their full telomere length when being continuously subcultured [47], which is linked to an increased risk of cancerogenicity [48]. In the same line of evidence must be seen the amount of cells needed for an effective tissue regeneration or to correct an urgently needed protein. For instance, the treatment of monogenetic disorders or acute forms of liver diseases can only be successfully achieved

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Langenbecks Arch Surg (2009) 394:985997 Good acceptance of cells in 2 patients/1 child died (follow-up from acute liver failure) Reduced phototherapy time and substantially decrease in post-transplant serum bilirubin Good liver function under continuous immunosuppression (2 years) Periods of hyperammonemia and clinically relevant crises could be reduced (13 months)/one child died after 3 months from a fatal metabolic decompensation Urea cycle disorders (3) Crigler-Najjar type I Crigler-Najjar type I Urea cycle disorders (4) 2008 2008 2005 2009 [113] [114] [115] [116] Female Male

hepatocytes in clinical studies were delivered into the splenic artery and not into the splenic pulp. This view is supported by Nagata and colleagues, who have shown that the route of hepatocyte delivery influences hepatocyte engraftment and function [18, 53]. Another open question remains as to whether the human spleen is capable of accommodating a sufficient number of functional hepatocytes to compensate for the cirrhotic liver. From the immunological point of view, the spleen represents the lions den, where transplanted cells could possibly cause greater immune response than in most other ectopic transplantation sites. Because of its large capacity and accessibility, the peritoneal cavity represents an alternative site for the cell therapy of end-stage liver disease. However, since hepatocyte suspensions do not survive for longer periods, only short-term effects may be achieved. Application of cells in patients with decompensated chronic liver disease has resulted in some improvement of laboratory parameters but was not able to change the natural cause of the disease (summary in Table 1). Encapsulation of hepatocytes [56, 57] or their attachment to micro-carriers may be an alternative method to improve efficacy of intraperitoneal cell transplantation. Implantable hepatocyte-based devices may represent another alternative for the treatment of end-stage liver disease [58]. However, these approaches are conceptual and still far from clinical application. The main advantage of cell therapeutic approaches to treat liver disease is that transplantation of cells is less invasive than whole-organ transplantation and can be performed repeatedly. However, one major limitation is the availability of human hepatocytes. A wider use of these techniques will not be possible until adequate numbers of cells for transplantation become more readily available. There are at least two possible sources that could meet the needs for transplantation, namely stem and precursor cellderived hepatocyte-like cells [4, 5, 5968] or reversibly replicating hepatocyte cell lines [69]. In recent years, numerous articles have reported about the generation of liver cells or hepatocyte-like cells from different types of extrahepatic stem or precursor. Ruhnke et al. described the gain of hepatocyte-like and islet cell-like features when peripheral blood monocytes were cultures in the presence of IL-3, M-CSF, and following FGF-4 [4, 5]. Tosh and coworkers could show hepatic features in pancreatic cells after treatment with dexamethasone [68, 70]. Jones et al. described hepatic differentiation of murine embryonic stem cells [63]. Sokal and coworkers could show hepatocyte-like features in differentiated fibroblasts [64], epithelial, and mesenchymal-like cells from the liver [66]. Further settings include differentiation of bone marrow cells [62], embryonic stem cells [63, 65, 67], and fat-derived mesenchymal stem cells [5961]. At first glance, this appears to provide exciting new opportunities for cell therapy, as some types

Outcome Age Table 1 Summary of hepatocyte transplantations performed Sex

1997 [107]

Crigler-Najjar type I Ornithine transcarbamylase deficiency

Glycogen storage disease type 1a Refsum disease Factor VII defficiency

1-antitrypsin deficiency

Recipient

1998 1999 2003 2002 2003 2004

Year

[108] [109] [110] [111] [51] [112]

Male Female Male Male Male Female Female Male Male

18 weeks 52 years 10 years 5 years 10 h 46 years 4 years 35 months 3 months 3 month to 3 years 8 years 9 years 13 years

Orthotopic liver transplantation (2 years) Orthotopic liver transplantation (4 years) Orthotopic liver transplantation (3.5 years) Normal ammonia level after 48 h died after 43 days Normal protein intake possible/orthotopic liver transplantation (6 months) Improved for 3 years Improved for 1 year Improved with decreased requirement for recombinant factor VII

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of stem cells proliferate efficiently in vitro and, therefore, may help to generate a larger supply of human hepatocytes or precursor cells for transplantation. Without doubt, the wide availability of human hepatocyte-like cells would be considered a major breakthrough and may open new perspectives for the treatment of liver disease. On the other hand, the results presented in these studies have to be carefully discussed. The results may be presented with farreaching conclusions with respect to the capacity of stem cell therapy, may have not been reproduced yet, or may have been interpreted in an overoptimistic manner. Therefore, there is a strong need for better characterization of generated human hepatocytes or precursor cells [71, 72]. Furthermore, homing of the transplanted cells has to be documented carefully to document possible engraftment sites. Moreover, there is still great potential in improving therapeutic success by optimizing the way of application and transplantation site.

Heart Stem cell treatment of the heart makes up an important field of research with significant clinical impact since cardiovascular disease is still the main cause of death worldwide. Although heart myocytes per se have regenerative potential [73], severe ischemic injury will rapidly lead to myocardial dysfunction due to inadequate self-regeneration capacity. Thus, treatment modalities for cardiac repair markedly supporting cardiomyocyte regeneration by rapid replacement of injured (non- or dysfunctional) cardiomyocytes gains increasing interest [74]. There have been a number of studies suggesting that transplantation of bone marrow stem cells is beneficial for myocardial repair and regeneration. Three weeks after injection of autologous bone marrowderived mononuclear cells into myocardial infarcted zone of swine, cardiac function, regional blood flow, and capillary density improved significantly [75]. In another study, only 9 days after injection of bone marrow cells from enhanced green fluorescent proteins (EGFP) transgenic male mice into the close surrounding of an ischemic area in Lin-c-kit+ female mice, resulted in the colonization of the infarcted area with male EGFP-positive cells expressing proteins characteristic of cardiac tissue, e.g., connexin43 [76]. Experimental studies with embryonic stem cells have well shown that implantation of these cells resulted in smaller infarct areas, improved left ventricular ejection fraction, and less extensive cardiac remodeling [2, 25]. Beside ethical issues, the clinical use of embryonic stem cells is not yet considered since long-term safety remains unclear. In this context, the formation of teratomas was described after cell transplantation into the brain [77]. Although no tumor occurrence was observed after trans-

plantation of mouse embryonic stem-cell-derived cardiomyocytes so far [25], the longest reported follow-up after transplantation of embryonic stem-cell-derived cardiomyocytes was only 32 weeks [78], highly suggesting that this issue still remains to be evaluated. In accordance to other organs, adult stem cells are also involved in cardiac regeneration [2, 74]. Adult stem cells that may be involved in cardiomyocyte regeneration can be divided broadly into three sources. Firstly, circulating cells, mainly of bone marrow origin that enter the circulation and home to the post-ischemic heart area; secondly, cells from noncardiac tissue such as bone marrow stem cells; and thirdly, resident cardiac stem cells [25, 74]. The existence of the later cell population has not yet been fully proven [79], and the main basis for its existence is an indirect conclusion. Despite intensive ongoing discussions on the impact and efficacy of the different (stem) cell types in cardiomyocyte differentiation, several clinical trials have been initiated based on promising experimental studies [80, 81]. At present, the heart is the organ in which most clinical experience with stem cell therapy is available [79]. Chronologically, skeletal myoblasts from the M. vastus lateralis were the first cells to be transplanted in a patient with severe ischemic heart failure in 2000 [82]. Feasibility of the procedure with recurrent injections of the final cell yield across the post-infarction scar as well as successful engraftment of these cells were well documented [83]; however, a strong pro-arrhythmic risk became apparent since the implanted myoblasts had different electrical properties and thereby facilitating micro-reentry circuits [84]. Consequently, all patients actuarially enrolled in the myoblast autologous grafting in ischemic cardiomyopathy trial received an internal cardioverter-defibrillator [85]. Clinical trials using bone marrow-derived cells have also reported to successfully improve patients outcome in terms of global and regional left ventricular function, tissue perfusion, and viability after acute myocardial infarction [86, 87]. However, treated patients in most studies were compared to historical controls or case-matched cohorts. Moreover, the nonuniform study criteria for the patient inclusion, the small patient numbers, and the variation in the selection of the injected cell type makes it difficult to draw an appropriate conclusion regarding the efficacy of bone marrow cell transplantation and cardiomyocyte regeneration [87]. So far, only few randomized and placebo-controlled clinical trials focusing on this issue are available [88]. Indeed, the technique of implanting bone marrowderived cells was feasible [87], but in contrast to experimental studies, obviously not dramatically effective in the clinical situation was achieved [88]. In the same line, it is now growing evidence that bone marrow cells may primarily secrete a wide variety of growths factors, thereby,

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enhancing angiogenesis rather than generating contractile cells [89]. Thus, further experimental studies are required to evaluate the exact mechanisms of myocardial regeneration and the relevance of cardiac stem cells [74]. Furthermore, two separate studies demonstrated that patients, receiving autologous bone marrow cells at the time of coronary artery bypass grafting, had improved perfusion of the infarcted myocardium [90]. There are studies in big animals that suggest very low numbers of injected cells that tend to disappear with time. Furthermore, if using undifferentiated stem cells, a risk remains when developing other types of tissue [91]. With this background, further studies are required to better characterize the phenotype and fate of injected cells. In conclusion, the promising experimental results of stem cell therapy in the heart have yet not been clinically confirmed, and further pathophysiological information on cardiomyocyte differentiation is highly required. In this context, Angelini et al. recently reported that many observers consider the scientific basis for stem cell therapy for the heart too primitive to justify clinical studies at present [79].

Kidney The adult human kidney contains at least 26 different cells types, such as tubular epithelial cells, glomerular cells, interstitial cells, and those of the vasculature. Recent reports suggest a role for stem cells, both from local and distant pools in normal turnover, e.g., to prevent chronic kidney diseases or in acute injury, e.g., following a toxic or ischemic event [92, 93]. In this context, bone marrowderived stem cells as well as embryonic and adult renal stem cells have been reported [92]. In contrast to embryonic stem cells, adult stem cells exist in various tissues and are able to maintain normal organ structure and function in both normal turnover and acute injury. So far, bone marrow-derived cells were thought to replace specialized adult renal cells as depicted from human and experimental studies. Thus, in clinical kidney transplantation, it was shown that bone marrow-derived cells of the recipient were found in renal vasculature and interstitium, renal tubules, and the glomerulus of the donor kidney [94]. Indeed, this finding was confirmed in several experimental studies in which bone marrow cells differentiated into proximal tubular epithelial cells, mesangial cells, endothelial cells, and interstitial cells [94]. Their ability to contribute to kidney tissues may also explain why bone marrow transplantation per se includes the risk of transmitting renal diseases. Indeed, it is now evident that bone marrow-derived cells can invade the kidney and may differentiate into renal cells and that acute renal injury followed by regeneration may increase their number [95].

Meanwhile, several studies have well underlined the substantial role of bone marrow-derived stem cells in both kidney maintenance [92] and repair after tissue injury. In this context, it was reported that bone marrow stem cells (marrow stromal cells) differentiated into renal tubular cells and blunted the rise of blood urea nitrogen in a mouse model of cisplatin injury [96]. Moreover, injected hematopoietic stem cells were shown to differentiate into renal tubular cells thereby improving kidney function in a mouse model of renal ischemia [97]. Most recently, adult stem cells originally from the kidney have been reported for the first time. Oliver et al. identified a population of slow-cycling cells residing in the mouse renal papilla that commence proliferating in response to ischemia/reperfusion injury and may migrate to the medulla [95]. Nevertheless, further characterization of this potential stem cell population is essential, and the existence of such an adult renal stem cell undoubtedly will have tremendous impact on the development of new therapies for acute and chronic kidney disease [98, 99]. So far, however, clinical studies with embryonic stem cells or adult renal stem cells are not considered. With respect to bone marrow-derived stem cells, it was demonstrated that these cells may engraft into different segments of the nephron, but human trials in renal diseases have yet not been started.

Cartilage and bone regeneration For centuries, physicians have been frustrated by the apparent lack to self-renewal of damaged cartilage. This problem has limited patients' complete recuperation from serious injuries involving knees, ankles, shoulders, and other major joints. Chondral lesions are commonly encountered in traumatology. These lesions can lead to premature osteoarthritis and may cause a decrease in quality of life with long-term costs of health care. The second serious problem in traumatology and orthopedic surgery is the recovery of large bone defects occurring after trauma or tumor. Such large defect can often be only insufficiently treated with conventional implants. Thus, these defects often cause loss of patients mobility and thereby loss of autonomy. Today, mainly autologous transplantation of osteochondral or cartilage tissue is used for treatment of cartilage defects and autologous spongiosa tissue for filling of bone defects. However, these sources are only limited, and obtainment of tissue is invasive and afflicted with risk of morbidity [100]. These techniques allow only recovery of relatively small defects in bone and cartilage. Today, great interest is aroused by stem-cell-based therapy of defects in the musculoskeletal system.

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Current concepts of treatment of cartilage defects include, among others, the recruitment of mesenchymal stem cells. Based on the knowledge of the (patho) physiology of articular cartilage, many clinicians have attempted to improve healing by drilling, abrasion, or micro-fracturing of the subchondral bone. The aim of these techniques is the recruitment of mesenchymal stem cells from the subchondral bone to stimulate the formation of cartilage repair tissue. However, this fibrocartilaginous repair tissue contains a high proportion of fibrous elements and is functionally inferior to normal hyaline cartilage. With these techniques, symptoms can be improved only temporarily. Therefore, improvements of this principle were performed in the last years like the matrix-coupled micro-fracturing. After micro-fracturing, the mesenchymal stem cells should migrate into the defect and differentiate from chondrocytes with the help of the collagenI/III-matrix (protection of cells and induction of differentiation). The short-term results are encouraging, but hyaline cartilage production was not demonstrated until now [101]. There have been several clinical studies in the last 30 years employing autologous mesenchymal stem cell injection in treatment of delayed or nonunion fracture healing of long bones [102] (see Table 2). Most of the studies were small observational phase I studies with short follow-up leaking control groups. Thus, most studies could not show clear advantages of stem cell application towards conventional treatment. However, these studies have shown that local application of bone-marrow-derived stem cells is rather safe, as no serious side effects were reported [103]. There are also several case reports demonstrating successful combination of mesenchymal stem cells with osteoconductive scaffolds leading to fusion of large bone defects and disappearance of cavitary defects [104]. These cases are promising, but systematical investigation of the beneficial effects of stem cell transplantation must follow.

Besides the use of mesenchymal stem cells and scaffolds, future concepts of treatment of bone and cartilage defects include the use of growth factors to promote proliferation and differentiation of the cells. These growth factors belong to the large group of cytokines that are produced by different cell types to attract mesenchymal stem cells to immigrate into the defect, proliferate, differentiate into osteoblasts or chondrocytes, and synthesize matrix. Studies showed that these factors increased the metabolism of osteoblasts and chondrocytes and promoted the healing of bone and cartilage defects. An important group of cytokines is the transforming growth factor (TGF) family with the bone morphogenetic proteins (BMPs). BMP-2 leads for example to an increase in proliferation of immigrating cells and differentiation of these to chondroblasts and an increase of the histological ICRS scores of cartilage regeneration in an animal trial. A sufficient production of the growth factor only in the defect area without influencing the surrounding tissue is important. Here, gene therapeutic procedures seem to be a promising tool in future. These could realize direct transfer of a therapeutic gene into cells of the defect. Carrier molecules that mediate this transfer process are named vectors. Fundamentally, one distinguishes two main groups: the nonviral (plasmids) and the viral vectors. The nonviral vectors possess a slight toxicity, are simply applicable, and do not show oncogenous potential [105]. The disadvantages exist in the slighter transduction rate and stability of the transfer. Viral transfer systems are, for example, retroviral [36], adenoviral, and lentiviral vectors with different operating mechanism. The advantage in contrast to nonviral systems is that the gene transfer is more stable and efficient. The disadvantages are especially for retroviral vectors the oncogenous potential. Newer vector systems are controllable [36], and the probability of insertional mutagenesis with a high tumor risk is unlikely [106]. After successful studies in animal models, these vector systems could become

Table 2 Clinical applications of mesenchymal stem cells performed in treatment of bone defects Author Salama1978 [102] Garg 1993 [117] Goel 2005 [103] Diagnosis Different bone defects Application Xenograft Bone marrow aspirate # patients Results 28 20 20 4 Results have been most satisfactory 17 out of 20 cases united in 5 (37)months 15 out of 20 patients showed radiologic bone union Follow-up to 7 years after surgery, good integration of Implant, no secondary fractures After 34.5 months, 95.1% cases had good spinal fusion results

Nonunion in long bones Percutanious injection of 15-20 ml bone marrow; 2times with interval of 3 weeks Tibial non-union Percutanious injection up to 15 ml bone marrow Isolated patients MSCs were expanded in vitro and seeded on hydroxyapatite scaffolds Enriched, autologous mesenchymal stem cells seeded on hydroxylapatite scaffold

Marcacci 2007 [104] Large bone diaphysis defect Gan 2008 [118] Dorsal spine fusion

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Langenbecks Arch Surg (2009) 394:985997 8. Yu J, Vodyanik MA, Smuga-Otto K et al (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science 318:19171920 9. Wernig M, Meissner A, Foreman R et al (2007) In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448:318324 10. Lowry WE, Richter L, Yachechko R et al (2008) Generation of human induced pluripotent stem cells from dermal fibroblasts. Proc Natl Acad Sci U S A 105:28832888 11. Park IH, Zhao R, West JA et al (2008) Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451:141146 12. Kim JB, Zaehres H, Wu G et al (2008) Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors. Nature 454:646650 13. Carey BW, Markoulaki S, Hanna J et al (2009) Reprogramming of murine and human somatic cells using a single polycistronic vector. Proc Natl Acad Sci U S A 106:157162 14. Li W, Wei W, Zhu S et al (2009) Generation of rat and human induced pluripotent stem cells by combining genetic reprogramming and chemical inhibitors. Cell Stem Cell 4:1619 15. Levesque JP, Hendy J, Winkler IG et al (2003) Granulocyte colony-stimulating factor induces the release in the bone marrow of proteases that cleave c-KIT receptor (CD117) from the surface of hematopoietic progenitor cells. Exp Hematol 31:109117 16. Petit I, Szyper-Kravitz M, Nagler A et al (2002) G-CSF induces stem cell mobilization by decreasing bone marrow SDF-1 and up-regulating CXCR4. Nat Immunol 3:687694 17. Francois S, Mouiseddine M, Mathieu N et al (2007) Human mesenchymal stem cells favour healing of the cutaneous radiation syndrome in a xenogenic transplant model. Ann Hematol 86:18 18. Gabelein G, Nussler AK, Morgott F et al (2008) Intrasplenic or subperitoneal hepatocyte transplantation to increase survival after surgically induced hepatic failure? Eur Surg Res 41:253 259 19. Harada K, Higaki S, Hashimoto K et al (2007) Study on the colonoscopic features of GVHD enteritis that developed after hematopoietic stem cell transplantation. Hepatogastroenterology 54:22212227 20. Hauger O, Frost EE, van Heeswijk R et al (2006) MR evaluation of the glomerular homing of magnetically labeled mesenchymal stem cells in a rat model of nephropathy. Radiology 238:200 210 21. Inagaki Y, Higashiyama R, Okazaki I (2007) Treatment strategy for liver fibrosis through recruitment and differentiation of bone marrow stem/progenitor cells. Hepatol Res 37:991993 22. Kawada H, Fujita J, Kinjo K et al (2004) Nonhematopoietic mesenchymal stem cells can be mobilized and differentiate into cardiomyocytes after myocardial infarction. Blood 104:3581 3587 23. Oyagi S, Hirose M, Kojima M et al (2006) Therapeutic effect of transplanting HGF-treated bone marrow mesenchymal cells into CCl4-injured rats. J Hepatol 44:742748 24. Terai S, Yamamoto N, Omori K et al (2002) A new cell therapy using bone marrow cells to repair damaged liver. J Gastroenterol 37(Suppl 14):162163 25. Van Laake LW, Van Hoof D, Mummery CL (2005) Cardiomyocytes derived from stem cells. Ann Med 37:499512 26. Glanemann M, Gaebelein G, Nussler N et al (2009) Transplantation of monocyte-derived hepatocyte-like cells (NeoHeps) improves survival in a model of acute liver failure. Ann Surg 249:149154 27. Horwitz EM, Gordon PL, Koo WK et al (2002) Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta:

interesting for the therapy of osteochondral lesions in humans.

Conclusions/future outlook/concluding remarks Although there are already a number of very interesting approaches to treat patients with stem cells under compassionate use, the authors believe that a registered clinical application based on stem cell technology will take at least an additional 5 to 10 years. Nevertheless, based on the tremendous medical need, it seems indisputably necessary to go new ways in research to overcome the lack of treatment options for devastating and degenerating diseases such as multiple scleroses or Alzheimer disease or to treat millions of patients suffering from hepatitis B or C virusinduced liver cirrhosis or to treat large bone and cartilage defects. Therefore, it seems to be a must to control the risk of tumorigenicity of a potential stem cell before this cell type can be used in clinic applications. Based on recent findings, the use of adult stem cells seems to be a more realistic option to find its way into clinical settings. The socalled iPS cells may overcome the dilemma of ethical issues raised by embryonic stem cells but may raise others. It is also clear that the development and application is not a domain solely managed by surgeons. Only an interdisciplinary approach will guarantee the most success to achieve a clinical approach.

Acknowledgements This work was partially supported by a grant of the BMBF (FKZ 01GN0984).

References
1. Thomson JA, Itskovitz-Eldor J, Shapiro SS et al (1998) Embryonic stem cell lines derived from human blastocysts. Science 282:11451147 2. Rubart M, Field LJ (2006) Cardiac regeneration: repopulating the heart. Annu Rev Physiol 68:2949 3. Chen N, Hudson JE, Walczak P et al (2005) Human umbilical cord blood progenitors: the potential of these hematopoietic cells to become neural. Stem Cells 23:15601570 4. Ruhnke M, Nussler AK, Ungefroren H et al (2005) Human monocyte-derived neohepatocytes: a promising alternative to primary human hepatocytes for autologous cell therapy. Transplantation 79:10971103 5. Ruhnke M, Ungefroren H, Nussler A et al (2005) Differentiation of in vitro-modified human peripheral blood monocytes into hepatocyte-like and pancreatic islet-like cells. Gastroenterology 128:17741786 6. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663676 7. Okita K, Ichisaka T, Yamanaka S (2007) Generation of germlinecompetent induced pluripotent stem cells. Nature 448:313317

Langenbecks Arch Surg (2009) 394:985997 Implications for cell therapy of bone. Proc Natl Acad Sci U S A 99:89328937 Lazarus HM, Koc ON, Devine SM et al (2005) Cotransplantation of HLA-identical sibling culture-expanded mesenchymal stem cells and hematopoietic stem cells in hematologic malignancy patients. Biol Blood Marrow Transplant 11:389398 Hidaka M, Su GN, Chen JK et al (2007) Transplantation of engineered bone tissue using a rotary three-dimensional culture system. In Vitro Cell Dev Biol Anim 43:4958 Marra KG, Defail AJ, Clavijo-Alvarez JA et al (2008) FGF-2 enhances vascularization for adipose tissue engineering. Plast Reconstr Surg 121:11531164 He L, Nan X, Wang Y et al (2007) Full-thickness tissue engineered skin constructed with autogenic bone marrow mesenchymal stem cells. Sci China C Life Sci 50:429437 Ma PX (2008) Biomimetic materials for tissue engineering. Adv Drug Deliv Rev 60:184198 Bosetti M, Santin M, Lloyd AW et al (2007) Cell behaviour on phospholipids-coated surfaces. J Mater Sci Mater Med 18:611 617 Liu X, Won Y, Ma PX (2005) Surface modification of interconnected porous scaffolds. J Biomed Mater Res A 74:84 91 Tachibana A, Nishikawa Y, Nishino M et al (2006) Modified keratin sponge: binding of bone morphogenetic protein-2 and osteoblast differentiation. J Biosci Bioeng 102:425429 Ueblacker P, Wagner B, Vogt S et al (2007) In vivo analysis of retroviral gene transfer to chondrocytes within collagen scaffolds for the treatment of osteochondral defects. Biomaterials 28:44804487 Lucke M, Wildemann B, Sadoni S et al (2005) Systemic versus local application of gentamicin in prophylaxis of implant-related osteomyelitis in a rat model. Bone 36:770778 Schmidmaier G, Wildemann B, Bail H et al (2001) Local application of growth factors (insulin-like growth factor-1 and transforming growth factor-beta1) from a biodegradable poly(D, L-lactide) coating of osteosynthetic implants accelerates fracture healing in rats. Bone 28:341350 Ameen C, Strehl R, Bjorquist P et al (2008) Human embryonic stem cells: current technologies and emerging industrial applications. Crit Rev Oncol Hematol 65:5480 Jensen J, Hyllner J, Bjorquist P (2009) Human embryonic stem cell technologies and drug discovery. J Cell Physiol 219:513 519 Molne J, Bjorquist P, Andersson K et al (2008) Blood group ABO antigen expression in human embryonic stem cells and in differentiated hepatocyte- and cardiomyocyte-like cells. Transplantation 86:14071413 Soderdahl T, Kuppers-Munther B, Heins N et al (2007) Glutathione transferases in hepatocyte-like cells derived from human embryonic stem cells. Toxicol In Vitro 21:929937 Sartipy P, Bjorquist P, Strehl R et al (2007) The application of human embryonic stem cell technologies to drug discovery. Drug Discov Today 12:688699 Ehnert S, Nussler AK, Lehmann A et al (2008) Blood monocytederived neohepatocytes as in vitro test system for drug metabolism. Drug Metab Dispos 36:19221929 Vigneau C, Zheng F, Polgar K et al (2006) Stem cells and kidney injury. Curr Opin Nephrol Hypertens 15:238244 Duan Y, Catana A, Meng Y et al (2007) Differentiation and enrichment of hepatocyte-like cells from human embryonic stem cells in vitro and in vivo. Stem Cells 25:30583068 Isaikina Y, Kustanovich A, Svirnovski A (2006) Growth kinetics and self-renewal of human mesenchymal stem cells derived from bone marrow of children with oncohematological diseases during expansion in vitro. Exp Oncol 28:146151

995 48. Stagg J (2008) Mesenchymal stem cells in cancer. Stem Cell Rev 4:119124 49. Strom S, Fisher R (2003) Hepatocyte transplantation: new possibilities for therapy. Gastroenterology 124:568571 50. Ott M, Schmidt HH, Cichon G et al (2000) Emerging therapies in hepatology: liver-directed gene transfer and hepatocyte transplantation. Cells Tissues Organs 167:8187 51. Sokal EM, Smets F, Bourgois A et al (2003) Hepatocyte transplantation in a 4-year-old girl with peroxisomal biogenesis disease: technique, safety, and metabolic follow-up. Transplantation 76:735738 52. Kobayashi N, Miyazaki M, Fukaya K et al (2000) Treatment of surgically induced acute liver failure with transplantation of highly differentiated immortalized human hepatocytes. Cell Transplant 9:733735 53. Nagata H, Ito M, Shirota C et al (2003) Route of hepatocyte delivery affects hepatocyte engraftment in the spleen. Transplantation 76:732734 54. Mito M, Kusano M, Kawaura Y (1992) Hepatocyte transplantation in man. Transplant Proc 24:30523053 55. Strom SC, Chowdhury JR, Fox IJ (1999) Hepatocyte transplantation for the treatment of human disease. Semin Liver Dis 19:3948 56. Aoki T, Koizumi T, Kobayashi Y et al (2005) A novel method of cryopreservation of rat and human hepatocytes by using encapsulation technique and possible use for cell transplantation. Cell Transplant 14:609620 57. Ringel M, von Mach MA, Santos R et al (2005) Hepatocytes cultured in alginate microspheres: an optimized technique to study enzyme induction. Toxicology 206:153167 58. Chan C, Berthiaume F, Nath BD et al (2004) Hepatic tissue engineering for adjunct and temporary liver support: critical technologies. Liver Transpl 10:13311342 59. Aurich H, Sgodda M, Kaltwasser P et al (2009) Hepatocyte differentiation of mesenchymal stem cells from human adipose tissue in vitro promotes hepatic integration in vivo. Gut 58:570 581 60. Aurich I, Mueller LP, Aurich H et al (2007) Functional integration of hepatocytes derived from human mesenchymal stem cells into mouse livers. Gut 56:405415 61. Banas A, Teratani T, Yamamoto Y et al (2007) Adipose tissuederived mesenchymal stem cells as a source of human hepatocytes. Hepatology 46:219228 62. Ishii K, Yoshida Y, Akechi Y et al (2008) Hepatic differentiation of human bone marrow-derived mesenchymal stem cells by tetracycline-regulated hepatocyte nuclear factor 3beta. Hepatology 48:597606 63. Jones EA, Tosh D, Wilson DI et al (2002) Hepatic differentiation of murine embryonic stem cells. Exp Cell Res 272:1522 64. Lysy PA, Smets F, Sibille C et al (2007) Human skin fibroblasts: From mesodermal to hepatocyte-like differentiation. Hepatology 46:15741585 65. Momose Y, Matsunaga T, Murai K et al (2009) Differentiation of monkey embryonic stem cells into hepatocytes and mRNA expression of cytochrome p450 enzymes responsible for drug metabolism: comparison of embryoid body formation conditions and matrices. Biol Pharm Bull 32:619626 66. Najimi M, Khuu DN, Lysy PA et al (2007) Adult-derived human liver mesenchymal-like cells as a potential progenitor reservoir of hepatocytes? Cell Transplant 16:717728 67. Ruhnke M, Ungefroren H, Zehle G et al (2003) Long-term culture and differentiation of rat embryonic stem cell-like cells into neuronal, glial, endothelial, and hepatic lineages. Stem Cells 21:428436 68. Shen CN, Slack JM, Tosh D (2000) Molecular basis of transdifferentiation of pancreas to liver. Nat Cell Biol 2:879887

28.

29.

30.

31.

32. 33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45. 46.

47.

996 69. Kobayashi N, Westerman KA, Tanaka N et al (2001) A reversibly immortalized human hepatocyte cell line as a source of hepatocyte-based biological support. Addict Biol 6:293300 70. Tosh D, Shen CN, Slack JM (2002) Differentiated properties of hepatocytes induced from pancreatic cells. Hepatology 36:534 543 71. Hengstler JG, Brulport M, Schormann W et al (2005) Generation of human hepatocytes by stem cell technology: definition of the hepatocyte. Expert Opin Drug Metab Toxicol 1:6174 72. Nussler A, Konig S, Ott M et al (2006) Present status and perspectives of cell-based therapies for liver diseases. J Hepatol 45:144159 73. Anversa P, Rota M, Urbanek K et al (2005) Myocardial aginga stem cell problem. Basic Res Cardiol 100:482493 74. Leri A, Kajstura J, Anversa P (2005) Cardiac stem cells and mechanisms of myocardial regeneration. Physiol Rev 85:1373 1416 75. Kamihata H, Matsubara H, Nishiue T et al (2001) Implantation of bone marrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands, and cytokines. Circulation 104:10461052 76. Orlic D, Kajstura J, Chimenti S et al (2001) Bone marrow cells regenerate infarcted myocardium. Nature 410:701705 77. Erdo F, Buhrle C, Blunk J et al (2003) Host-dependent tumorigenesis of embryonic stem cell transplantation in experimental stroke. J Cereb Blood Flow Metab 23:780785 78. Min JY, Yang Y, Converso KL et al (2002) Transplantation of embryonic stem cells improves cardiac function in postinfarcted rats. J Appl Physiol 92:288296 79. Angelini P, Markwald RR (2005) Stem cell treatment of the heart: a review of its current status on the brink of clinical experimentation. Tex Heart Inst J 32:479488 80. Segers VF, Lee RT (2008) Stem-cell therapy for cardiac disease. Nature 451:937942 81. Yousef M, Schannwell CM, Kostering M et al (2009) The BALANCE Study: clinical benefit and long-term outcome after intracoronary autologous bone marrow cell transplantation in patients with acute myocardial infarction. J Am Coll Cardiol 53:22622269 82. Menasche P, Hagege AA, Vilquin JT et al (2003) Autologous skeletal myoblast transplantation for severe postinfarction left ventricular dysfunction. J Am Coll Cardiol 41:10781083 83. Hagege AA, Carrion C, Menasche P et al (2003) Viability and differentiation of autologous skeletal myoblast grafts in ischaemic cardiomyopathy. Lancet 361:491492 84. Menasche P (2005) Stem cells for clinical use in cardiovascular medicine: current limitations and future perspectives. Thromb Haemost 94:697701 85. Menasche P, Alfieri O, Janssens S et al (2008) The Myoblast Autologous Grafting in Ischemic Cardiomyopathy (MAGIC) trial: first randomized placebo-controlled study of myoblast transplantation. Circulation 117:11891200 86. Tse HF, Kwong YL, Chan JK et al (2003) Angiogenesis in ischaemic myocardium by intramyocardial autologous bone marrow mononuclear cell implantation. Lancet 361:4749 87. Ye L, Haider H, Sim EK (2006) Adult stem cells for cardiac repair: a choice between skeletal myoblasts and bone marrow stem cells. Exp Biol Med (Maywood) 231:819 88. Cleland JG, Coletta AP, Abdellah AT et al (2007) Clinical trials update from the American Heart Association 2006: OAT, SALT 1 and 2, MAGIC, ABCD, PABA-CHF, IMPROVE-CHF, and percutaneous mitral annuloplasty. Eur J Heart Fail 9:9297 89. Murry CE, Soonpaa MH, Reinecke H et al (2004) Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts. Nature 428:664668

Langenbecks Arch Surg (2009) 394:985997 90. Stamm C, Westphal B, Kleine HD et al (2003) Autologous bonemarrow stem-cell transplantation for myocardial regeneration. Lancet 361:4546 91. Laham RJ, Oettgen P (2003) Bone marrow transplantation for the heart: fact or fiction? Lancet 361:1112 92. Brodie JC, Humes HD (2005) Stem cell approaches for the treatment of renal failure. Pharmacol Rev 57:299313 93. Lin F (2006) Stem cells in kidney regeneration following acute renal injury. Pediatr Res 59:74R78R 94. Ricardo SD, Deane JA (2005) Adult stem cells in renal injury and repair. Nephrology (Carlton) 10:276282 95. Oliver JA, Maarouf O, Cheema FH et al (2004) The renal papilla is a niche for adult kidney stem cells. J Clin Invest 114:795804 96. Morigi M, Imberti B, Zoja C et al (2004) Mesenchymal stem cells are renotropic, helping to repair the kidney and improve function in acute renal failure. J Am Soc Nephrol 15:17941804 97. Kale S, Karihaloo A, Clark PR et al (2003) Bone marrow stem cells contribute to repair of the ischemically injured renal tubule. J Clin Invest 112:4249 98. Anglani F, Forino M, Del Prete D et al (2004) In search of adult renal stem cells. J Cell Mol Med 8:474487 99. Bates CM, Lin F (2005) Future strategies in the treatment of acute renal failure: growth factors, stem cells, and other novel therapies. Curr Opin Pediatr 17:215220 100. Hangody L, Vasarhelyi G, Hangody LR et al (2008) Autologous osteochondral graftingtechnique and long-term results. Injury 39(Suppl 1):S32S39 101. Steinwachs MR, Guggi T, Kreuz PC (2008) Marrow stimulation techniques. Injury 39(Suppl 1):S26S31 102. Salama R, Weissman SL (1978) The clinical use of combined xenografts of bone and autologous red marrow. A preliminary report. J Bone Joint Surg Br 60:111115 103. Goel A, Sangwan SS, Siwach RC et al (2005) Percutaneous bone marrow grafting for the treatment of tibial non-union. Injury 36:203206 104. Marcacci M, Kon E, Moukhachev V et al (2007) Stem cells associated with macroporous bioceramics for long bone repair: 6- to 7-year outcome of a pilot clinical study. Tissue Eng 13:947955 105. Raty JK, Lesch HP, Wirth T et al (2008) Improving safety of gene therapy. Curr Drug Saf 3:4653 106. Yi Y, Hahm SH, Lee KH (2005) Retroviral gene therapy: safety issues and possible solutions. Curr Gene Ther 5:2535 107. Strom SC, Fisher RA, Thompson MT et al (1997) Hepatocyte transplantation as a bridge to orthotopic liver transplantation in terminal liver failure. Transplantation 63:559569 108. Fox IJ, Chowdhury JR, Kaufman SS et al (1998) Treatment of the Crigler-Najjar syndrome type I with hepatocyte transplantation. N Engl J Med 338:14221426 109. Bohnen NI, Charron M, Reyes J et al (2000) Use of indium-111labeled hepatocytes to determine the biodistribution of transplanted hepatocytes through portal vein infusion. Clin Nucl Med 25:447450 110. Horslen SP, McCowan TC, Goertzen TC et al (2003) Isolated hepatocyte transplantation in an infant with a severe urea cycle disorder. Pediatrics 111:12621267 111. Muraca M, Gerunda G, Neri D et al (2002) Hepatocyte transplantation as a treatment for glycogen storage disease type 1a. Lancet 359:317318 112. Dhawan A, Mitry RR, Hughes RD et al (2004) Hepatocyte transplantation for inherited factor VII deficiency. Transplantation 78:18121814 113. Meyburg J, Hoerster F, Weitz J et al (2008) Use of the middle colic vein for liver cell transplantation in infants and small children. Transplant Proc 40:936937

Langenbecks Arch Surg (2009) 394:985997 114. Allen KJ, Mifsud NA, Williamson R et al (2008) Cell-mediated rejection results in allograft loss after liver cell transplantation. Liver Transpl 14:688694 115. Ambrosino G, Varotto S, Strom SC et al (2005) Isolated hepatocyte transplantation for Crigler-Najjar syndrome type 1. Cell Transplant 14:151157 116. Meyburg J, Das AM, Hoerster F et al (2009) One liver for four children: first clinical series of liver cell transplantation

997 for severe neonatal urea cycle defects. Transplantation 87:636 641 117. Garg NK, Gaur S, Sharma S (1993) Percutaneous autogenous bone marrow grafting in 20 cases of ununited fracture. Acta Orthop Scand 64:671672 118. Gan Y, Dai K, Zhang P et al (2008) The clinical use of enriched bone marrow stem cells combined with porous beta-tricalcium phosphate in posterior spinal fusion. Biomaterials 29:39733982